Table of contents : 

Histochemistry Microscopy Cell conservation Purification of cell types Cell culture Biochemical testings Pharmacology Immunoassays Clonality assays

Nucleic acid extraction Imaging Transfection of ... nucleic acids aims proteins every kind of molecule Bibliography

• Cell conservation
• cryogenics
• dry ice (solid CO₂) : -78 °C
• liquid N₂ : -196 °C
Additives : dimethylsulphoxide (DMSO), glycerol, Suc, ...
• cooling of blood platelets clusters the von Willebrand factor (vWF) receptor complex. Macrophage a_Mb₂ integrins bind to the GPIb (a + b) subunit of the clustered complex, resulting in rapid clearance of transfused, cooled platelets. This precludes refrigeration of platelets for transfusion, but the current practice of room temperature storage has major drawbacks. We document that a_Mb₂ is a lectin that recognizes exposed ß-N-acetylglucosamine residues of N-linked glycans on GPIb. Enzymatic galactosylation of chilled platelets blocks a_Mb₂ recognition, prolonging the circulation of functional cooled platelets. Platelet-associated galactosyltransferase produces efficient galactosylation when UDP-galactose is added, affording a potentially simple method for storing platelets in the cold. Transferring the technique to stem cells is tricky because, unlike platelets, they have nuclei that must be perfectly preserved.
Today researchers save stem cells by freezing them in expensive vats of liquid nitrogen at -140 ºC. To stop damaging ice crystals forming inside the cells, they bathe them in a toxic chemical such as DMSO. This must be washed off before cells can be used.
Web resources : dmoz Open Directory Project : Cryobiology
• liophilization / cryodrying : high-speed freezing (to create only small-sized H₂O crystals) followed by transition to gaseous state. A buffer is needed to mantain A_w = 1%.
• air drying : several scientists are looking for a way to air-dry cells, comparable to the technique that turns grapes to raisins. Dried stem cells might be stored in portable packets that can be ripped open and their contents mixed with water at the scene of an accident. After drying and soaking the stem cells in the anti-freeze sugar trehalose (which sloshes around in organisms that survive dehydration, such as brine shrimp, baker's yeast and certain drought-resistant resurrection plants) and arbutin, < 40% of the cells was water. When rehydrated immediately afterwards, up to half grew and divided and cells can be revived after several weeks on the shelf.. Dehydration must remove as much water as possible so that cells' metabolism is halted, otherwise they churn out waste that poisons their surroundings. But cell membranes crack if they are overly parched. Researchers are experimenting to strike the right balance.
• Purification of cell types
• tissue microdissection : dissection of tissue or cells under the microscope.
• laser-capture microdissection (LCM) : energy from a low-power laser fitted to an inverted microscope is used to melt a thin vinyl film to precise loctions on a tissue section to bind targeted cells. After the appropriate cells have been selected, the film and adherent cells are removed for gene-expression studies.
• magnetic activated cell sorter (MACS) : immunomagnetic microbeads [movie]
• cytometry : the characterization and measurement of cells and cellular constituents.
• microfluorometry / cytophotometry : the study of organic compounds within cells by means of the cytophotometer (a photometer for measuring localization of organic compounds within cells by measuring the light intensity through selected stained areas of cytoplasm)
• image cytometry : a technique in which histologically prepared cells are imaged using a scanning technique that divides the whole image into many smaller elements and aspects, which can then be analyzed by a computer and compared between many different cells in a series.
• flow cytometry : a technique in which cells suspended in a fluid flow one at a time through a focus of exciting light, which is scattered in patterns characteristic to the cells and their components; they are often labeled with fluorescent markers so that light is first absorbed and then emitted at altered frequencies. A sensor detecting the scattered or emitted light measures the size and molecular characteristics of individual cells; tens of thousands of cells can be examined per minute and the data gathered are processed by computer.

The cell structure(s)/molecule(s) are marked with
• a fluorophore (direct ligand)
• enzyme substrate
• nucleic acid binder
• a fluorophore-conjugated ligand
• monoclonal antibodies (immunophenotyping) : pelletize antigen-bearing cells (5 x 10⁶ each in 3 wells : the first with no antibody added; the second with only secondary antibody added; and the third with both immune serum and secondary antibody added) by centrifuging 3 minutes at 1,600 rpm; add 100 mL of FACS buffer (PBS 3%, 0.2 NaN₃) per well and pelletize again; add 1 mL/well of serum from surviving animals to the second and third wells to obtain a final dilution = 1 : 200. Incubate for 20' over ice (covered with aluminum paper) and pelletize again. Washing with 100 mL of FACS buffer. Add 1 mL of secondary goat anti-mouse mAb to the third well only. Incubate for 20 minutes and pelletize again. Add 100 mL of FACS buffer and read at FACS (after usage clean the FACS with hypochlorite first and water then)
• lectins
• cytokines
• nucleic acid probe
• beads or particles
... and the unbound is washed out (or, in the case of substrates, maintains another excitation peak). All fluorophores need to be stimulated from the same laser beam, and hence need a common (or at least similar) excitation spectrum. Stokes's shift : l_excitation - l_emission (nm).
A cytometer consists of :
• optics
• necessary for excitation
• light source(s), usually laser(s)
• argon ion lasers => 488 nm (the absorption peak of several commonly used fluorochromes and dyes)
• helium-neon lasers (He-Ne) lasers => 633 nm
• helium-cadmium lasers (He-Cd) => 325 nm
• red diode lasers => 635 nm
• focusing lens
• necessary for collection of the light emitted from, or scattered by, the cells.
• detection and quantitation of laser light scattered at
• 90° : orthogonal or side scatter (SS), correlated with the granularity of the cell
• 180°C : forward scatter (FS), correlated with cell size
• detection and quantitation of as amany as 11 colors of  immunofluorescence emanated at 90°
• if the fluorescent light is separate as a result of sequential laser intercepts, then the signals may be sent in dfferent directions through the use of
• beam splitter
• half mirrors
• if all fluorescent light is mixed
• dichroic mirrors reflect light above a specified wavelength and permit light below that wavelength to pass through
• short pass filters absorb light above a specified wavelength and permit only light below a specified wavelength to pass through
• long pass filters absorb light below a specified wavelength and permit only light above a specified wavelength to pass through
• bandpass filters permit only light in a specified range of wavelenght to pass through, absorbing light with wavelength above and below that range
• 488 BP filter
• fluidics : coaxial stream whose pressure is controlled by electronics.
• inner sample stream : at least 1,000 up to 100,000 cells per second
• outer sheath stream to focus the inner stream and to minimize the turbulence arising from the resistance of the tubing, allowing cells in the sample stream to be presented to the laser one at a time and in a sequence that can be maintained for interrogation by multiple lasers.
Although most clinical assays do not use sorting (fluorescence activated cell sorter (FACS)), it is commonly used in research. For sorting, the coaxial stream is broken into droplets after interrogation by the laser, and these droplets are then charged according to the fluorescence of the cell (or cells) contained within the droplet. The drops are then sorted by charged deflection plates (controlled by electronics) that alter the path of the drop as a result of the charge. Some cytometers sort cells in 4 directions.
The liquid waste is transported by a vacuum into a tank which should contain a disinfectant such as bleach to minimize biohazard risk and must be manually emptied at periodic intervals.
• electronics
• photomultiplier tube (PMT) / photodiodes convert the incident light into electronic pulses. The sensitivity of the PMTs to light is controlled by the voltage supplied to the PMT, thus electronics are needed to control this voltage
• signal amplification
• analogue-to-digital converters (ADCs) for computer analysis
• compensation circuits controlled by electronics
• computer (via Institute for Electronic and Electrical Engineers (IEEE) port) : CellQuest™ software
• controlling instrument operation
• analysis of data
• presentation of data
• 2-parameter histograms
• dot plots
• export statistics for use in spreadsheets, used for further analysis and graphics
• storage of data in listmode files (usually on CD-ROMs due to lack of high capacity storage devices)
• algorithms used to compare data
• Kolmogorov-Smirnov (K-S) two sample algorithm is a commonly used method to determine the confidence interval (D value) with which one can make the assertion that 2 flow cytometric univariate histograms are different (P = 0.001)
• Overton cumulative histogram subtraction algorithm essentially subtracts histograms on a channel-by-channel basis to provide a percent of positive cells.
• super-enhanced Dmax subtraction (SED) is a new sophisticated algorithm by Bruce Bagwell to compute %Positives when comparing histograms.
• probability binning (PB) comparison is related to the Cox chi-square approach, but with modified binning such that it minimizes the maximal expected variance. This algorithm has been shown to detect small differences between 2 populations and it does so in a quantitative way.
Single cell analysis : if clumps or doublets causing misinterpretations (e.g. CD4⁺ cell + CD8⁺ cell = DP cell ; 2 nuclei = G₂/M cell) are known to exist in the specimen, they can be filtered out before analysis or gated out using the appropriate parameters.
Compensation to subtract the portion of the observed fluorescence resulting from spectral overlap :
• pre-data acquisition compensation
• visually determined (and manually set)
• established by software programs
• post-data acquisition compensation
• established by software programs
Sequential gating with Boolean logic :
• based on light scatter (traditional SS-to-FS plot). E.g. for peripheral blood :


• based on immunofluorescence
... performed
• during acquisition of data
• during analysis of listmode files : the gating is not permanent and list mode data may be regated an infinite number of times if the original file is not overwritten
Quantitative cytometry :
• express the detected fluorescence in molecules of equivalent soluble fluorescein (MESF units)
• quantitate antigen density in antibody binding capacity (ABCs) in immunophenotyping
[cells having the target molecule]_{sample volume} =
•  = [cells having the target molecule]%_{sample volume, detected with cytometer}  ^. absolute number of cells_{sample volume, performed manually or on other instrumentations}
•  = [fluorescent beads]_{sample volume, known} ^. [fluorescent beads]%_{sample volume, detected with cytometer} / [cells having the target molecule]%_{sample volume, detected with cytometer}. This method has higher accuracy and precision.
Quality control :
• of the flow cytometer : analyzing beads (or other particles) with known, stable, fluorescence and light scattering properties
• of the reagents and specimen preparation techniques : staining and analyzing normal control specimens and comparing the data to a historical database
BD Biosciences, a segment of Becton, Dickinson and Company (BD), developed one of the earliest commercial flow cytometers, called a fluorescence-activated cell sorter (BD FACS), in the early 1970s. For this reason, some people still use the term FACS generically to mean flow cytometry and the instruments that perform it. Today FSC and SSC are taken at breathtaking speeds exceeding 70,000 cells per second with unsurpassed accuracy and sensitivity. For this reason, flow cytometry is ideally suited for studies that need to distinguish among up to several million cells based on many different parameters. The recent marriage of flow cytometers with other detection systems, such as imaging devices and multiplex bead-based assays, has greatly enhanced the amount of information that can be gathered from each cell. A major development in flow cytometry has been the availability of an arsenal of fluorescent probes. In addition, it is now possible to more precisely quantify the amount of signal coming from one cell relative to another. E.g. researchers can identify receptors on the surface of cells, total DNA, the amount of mRNA for a particular gene, calcium concentrations, membrane potential and pH. Some of the newer probes allow researchers to measure the amount of protein phosphorylation in a cell to study signaling cascades. BD Biosciences alone sells approximately 25 types of fluorescent reporter dyes coupled to antibodies, probes and other reagents. To take advantage of the growing number of fluorescent reagents, flow cytometers have multiple lasers to stimulate different excitation wavelengths and several detectors to collect different color signals. The top-of-the-line machine produced by BD Biosciences can detect up to 18 colors. Germany-based Partec, which started distributing flow cytometers in 1969, offers different configurations of instruments and lasers to suit customers' needs.There is very little limitation for detecting more colors on the instrument side. But despite the boundless possibilities, only a handful of published studies look at > 8 colors simultaneously and most will use 4-5. Complex multicolor analyses are difficult to conduct : one of the main stumbling blocks is the data processing and proper interpretation
• if you have ever had to do it, you know the trials and tribulations associated with generating a stable cell line that expresses your favorite gene. Chromocell, a startup company based in New Jersey, has found a way to deliver stable cell lines that reproducibly and reliably express high levels of functional proteins with greater ease and efficiency. The technology uses a combination of special fluorescent probes to detect the mRNA corresponding to the gene of interest in individual transfected cells and high-speed sorting of the target cells by flow cytometry. When the probes are added to a batch of cells, only those cells that contain the target message end up with a fluorescent signal. The cytometer is then programmed to select cells with the highest amount of signal so that they can be further studied to determine whether the expressed protein is functional. For proteins made up of more than one subunit, cells are transfected with, for example, 3 individual genes and the cytometer simultaneously tests for the presence of all 3 signals in a cell. The procedure is largely automated making it possible to screen millions of cells in a matter of minutes. When you get so many cell lines that express the gene you are interested in, you can then select the cells that meet other criteria, for example stability. In addition to stability, the researchers test the response to different treatments to see whether the expressed protein behaves as it should. Another advantage of the technology is that unlike high-throughput methods for cell selection in which transfected genes carry some kind of fluorescent tag, such as a GFP molecule, the expressed protein is as close to the native configuration as possible. The company, which was established in 2002 by researchers from Günter Blobel's lab at The Rockefeller University, produces stable cell lines as a service and is now exploring other applications of the technology, in particular for antibody biologics production
• every flow cytometer includes software for collecting and analyzing information gathered during an experiment. The analysis typically involves finding out how many cells from the sampled population meet a criteria of interest, and the data can be displayed in a number of different formats, most frequently dot plots or histograms. Basically, histograms quantitate intensities of scatter or fluorescence based on one parameter, whereas dot plots quantitate percentages of cells with various properties. Dot plots are typically used to detect small numbers of cells that are well separated from the main populations of the cells present. In addition, in the software packages that come with some imaging cytometers, such as Amnis' IDEAS data analysis software, the dot plots are linked to images of the cells. That means that a user can click on an individual dot to see the corresponding image or can draw a 'gate' around a population of dots to see what that cells that fall within the gate look like. This is very useful for confirming that the dots really do represent the cells you think they do and aren't clumps of cells or other artefacts. Many companies also sell software packages separately from the instrumentation. One of the most popular products is Tree Star's FlowJo software, which can analyze data from any flow cytometer and will work in both a Windows and MacIntosh environment. FlowJo is based around 'workspace' documents—similar to pages in a notebook—that contain information about the samples, along with all the gates, graphs, statistics, tables and reports applied to the data. FlowJo provides several different analysis platforms for standard analyses such as gating and statistics, as well as more special ones, such as DNA content, cell cycle analysis, kinetics, proliferation, calibration and statistical comparisons. Other examples include BD CellQuest, a popular commercial flow cytometry data acquisition and analysis software for the MacIntosh computer produced by BD Biosciences. FSC Express from De Novo Software is a very advanced analysis product that runs on the PC. Verity has Winlist, a comprehensive data analysis package for the PC, and Modfit, one of the main DNA and cell cycle analysis packages. Some software packages are geared toward more sophisticated applications. Applied Cytometry Systems (ACS) has developed the StarStation software to drive the Luminex flow cytometer and help analyze the data generated from it. Bio-Rad and MiraiBio have similar software products called Bio-Plex Manager and MasterPlex, respectively. The software takes care of calibrating the instrument, running the assays and collecting the data. In September 2005, ACS will release a new product that reads files generated in StarStation and analyses them in custom ways according to a particular plug in application. The first plug in, called SNP-R, will be used to compare a pair of alleles to determine which one is present in a given sample. It is similar to data obtained using a SNP microarray but different in that the sample is in flow. The company plans to continue developing different types of plug-ins for customers.
Improved lasers have not only given way to greater accuracy and sensitivity, but also smaller instruments. Lasers are becoming smaller and cheaper and so are the instruments. In the past 3 decades, high-end machines have gotten smaller without compromising power. DakoCytomation's CyAn cytometer contains 3 lasers (blue, red and violet), and can measure forward and side scatter and up to 9 colors of fluorescence, all at speeds of over 30,000 cells per second. According to the company, the instrument is 6-10 times smaller than earlier models with comparable capabilities. BD Biosciences sells a low-cost benchtop analyzer called BD FACSArray. With 2 lasers, it can measure 15,000 cells per second and visualize 4 colors, as well as cell size and heterogeneity. In addition, several companies, including Guava Technologies, Beckman Coulter and Partec, sell small 2-color units that are more targeted to specific applications, such as measuring cell proliferation or apoptosis. Customers like the fact that they use their budget for the configurations that they need. The most recent newcomer in this arena is Beckman Coulter's Cell Lab Quanta flow cytometry system, which began shipping in April of 2005. Built on a joint development effort with NPE Systems, the Quanta combines Coulter volume measurement (an electrical sensing method for counting and sizing cells), with 2-color fluorescence capabilities. In September 2005, the company will release a new model that can also measure light scatter and one additional color. The size and affordability of these smaller instruments, as well as the ease of use, have made it possible for them to be used in developing countries to monitor HIV infection in the population.
• flow cytometry was thrust into the spotlight by the AIDS epidemic in the 1980s as it provides a quick and accurate way to count CD4⁺ and CD8⁺ T cells in HIV-infected patients. Until recently flow cytometers and the necessary reagents were too expensive and too difficult to implement in developing countries—where HIV infection is ravaging the population. Thus, the development of smaller and more affordable, robust instruments has had a substantial impact in this part of the world.  Partec has 2 kinds of mobile flow cytometry units: one, housed in a car, which is powered by the car battery and solar panels, and the other, part of a laboratory truck, is powered by its own generator. Since 2002, > 200 CyFlow instruments have been placed in Sub-Saharan Africa, offering CD4 counting at a cost of about $2.2. In addition to CD4 counts, the Partec instruments measure the number of CD4⁺ T cells as a % of the total number of lymphocytes. Recent studies have revealed that in infants, CD4 counts normally decrease with age, whereas the proportion of CD4 remains stable. This means that the threshold of 200 CD4⁺ T cells generally used to decide when to start antiviral therapy cannot be safely used for children. The decision to start therapy in pediatric patients should instead be based on the CD4 % value. Guava Technologies' EasyCD4 system weighs a mere 35 pounds. These systems and optimized CD4 assay kits have been sold throughout Africa, India and southeast Asia for CD4 counting at slightly > $1 per test. Other companies offering flow cytometry products for CD4 counting in developing countries include BD Biosciences and Beckman Coulter.
Some flow cytometers can physically separate a group of cells from the rest. The instrument puts a negative or positive charge on specific cells that have defined properties, and then deflects these charged cells to separate collection tubes. Because multiple fluorochromes can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of several markers—a capability that is increasingly exploited in stem cell research protocols. As a recent example, Sean Morrison's group at The University of Michigan, Ann Arbor, was able to isolate mouse HSCs of unprecedented purity from blood, based on the combined expression of the receptors of the SLAM family^ref. The ability to do these kinds of experiments rests partly on improved sensitivity of detection. But another requirement for cell sorting, especially when dealing with a population of rare cells, is speed. DakoCytomation's MoFlo was the first true high-speed cell sorter and the first to allow separation of four different cell populations simultaneously. It has high performance in terms of yield, cell viability and purity, and runs at very high speed, reaching up to 70,000 cells per second."The sorter has an open design, which means that the user can add different kinds of lasers as new fluorechromes are developed. Another popular cytometer with cell sorting capability is BD Biosciences' FACSVantage SE System. With a new generation of lasers and advanced optics, the company claims it provides enormous flexibility and sensitivity. It sorts up to 4 populations of cells simultaneously at rate of 16,000 cells per second, with sorted fractions exhibiting > 99% purity. Traditionally, flow cytometers with cell sorting capability were large instruments, but that is no longer the case. In April 2003, BD Biosciences began shipping the first high-performance benchtop cytometer with electrical sorting capabilities and, according to the company, the instrument, called BD FACSAria, has taken the market by storm. Improvements in fluidics and optics allow for higher speed sorting (25,000 events per second) with less stress on the sorted cells and enhanced overall sensitivity. The system uses 3 lasers to allow detection of 13 colors as well as scatter. An exciting advance has been the combination of flow cytometry with imaging tools and devices. Amnis Corporation pioneered multispectral image flow cytometry, a method that allows individual cells to be visualized as they zoom by the laser beam. Amnis' ImageStream 100 is similar to a flow cytometer with the important distinction that the traditional detectors have been replaced by a sensitive CCD camera, custom designed by the company. The camera takes as many as 6 independent images (brightfield, darkfield and up to four fluorescent images) of each cell at a rate of about 100 cells per second. Although the speed is slow for a flow cytometer, it beats that of any high-content screening system hands down. Because it can image a large number of cells at once, the ImageStream is ideally suited to gather a statistically meaningful picture of a population of cells. If there is any variability in a cellular response to a treatment, you will not get the necessary statistical information using a microscope. The instrument provides the same measurements as a traditional flow cytometer but with the added benefits that you can also calculate where the fluorescent signal is coming from in the cell. For example, a researcher can measure the localization of 2 cell-surface markers relative to one another or determine the cytoplasmic versus nuclear location of a molecule, all in a large population of cells. Many researchers believe that tumors contain a rare population of therapy-resistant resting cells, which act like stem cells to give rise to cells that continuously divide. Laser scanning cytometers (LSC) are also capable of visualizing the location of fluorescent signals in a cell, but using a different strategy. In an LSC the sample does not move, rather the laser beam sweeps over the cells to construct an image in a manner similar to confocal microscopy. Unlike a microscope, the LSC allows the user to scan a relatively large area without the need to refocus. In addition, the LSC allows the quantification of all the fluorescent light emitted from the entire cell depth at each location in the specimen, just as a flow cytometer does. It has the same quantitative power as flow cytometry in terms of sensitivity and precision, but with the addition of imaging and the ability to work with live or fixed adherent cells, using a variety of carrier types. CompuCyte's LSC has 4 fluorescence and 2 scatter detectors, to capture up to 6 images of cells; the sample can be either in suspension, adhering to a slide or in tissue sections. The LSC is well-suited for applications that cannot be done in flow. It is good for studying apoptosis in adherent cells. Removing cells from a culture dish to suspend them can simulate cell death, so it is preferable to analyze them while they are still on their growth surface. CompuCyte's laser scanning cytometers can analyze both fluorescent and chromatic dyes, as well as combinations of the 2. This extends cytometric investigations to objective cytometric analysis of tissue sections without compromising tissue architecture and allows pathologists to view samples in the manner that they are accustomed to, coupled with additional quantitative data similar to flow cytometry report. In 2006, the company will provide an additional feature with their instruments to allow the measurement of decay times of fluorescent labels. Because conditions like pH, oxygen or cation concentrations affect the rate of decay of a fluorescent dye, decay times can provide a sensitive measure of the characteristics of a cell's microenvironment. Another LSC system, called SurroScan, was developed at SurroMed Inc. (which was acquired in early 2005 by Pharmaceutical Product Development Biomarker Discovery Sciences, a company that provides cytometry services for clinical studies and trials). The system combines the best features of a hematology analyzer and a flow cytometer. Samples are placed in a volumetric capillary array that holds 32 assays with 3-4 colors in each. As the laser scans over each capillary, the instrument gathers information to classify and quantify cells in blood or other fluids. Results include both absolute cell counts and relative levels of antigen expression. You can process a large number of assays on a large number of samples in a reproducible way. New systems using multiplex bead arrays push the potential applications for flow cytometry even further. These systems simultaneously measure up to 100 compounds in suspension using very small volumes. Common applications include profiling cytokine concentrations in serum or identifying which proteins are phosphorylated after activation of a given kinase. The flow cytometers and reagents developed by Luminex combine the power of flow cytometry with the simplicity of an ELISA plate reader. Instead of reading cell-based assays, they read a microsphere-based assays. The Luminex method uses microsphere sets carrying variable quantities of two different fluorescent dyes that produce up to 100 different shades of color. Each bead is coupled to a unique antibody or probe that recognizes a specific molecule. After the beads are mixed with a sample—for example, cell lysates—and added to the instrument, the unique color signature on each bead reveals the identity of the bound molecules. Several companies, including Applied Cytometry Systems, Biosource International, MiraiBio, Qiagen, Upstate, LINCO Research, Marligen Biosciences and others, are licensed to sell Luminex instruments or reagents. In addition, other companies have developed similar technologies. BD Biosciences' FACSArray analyzer can process bead-based assays using the company's own brand of reagents. Beckman Coulter also produces multiplex bead assays that, instead of having 2 colors on one bead, use 2 beads per assay. Although relatively new on the market, the power of this technology is clear. Todd Golub and Robert Horvitz used it to analyze the expression of microRNAs (miRNAs) in different human cancers^ref. As miRNAs are only about 21 nucleotides long and share similar sequences^ref, it is difficult to detect specific miRNAs using glass slide microarrays—a popular method for expression profiling. To overcome the problem, the researchers coupled oligonucleotide probes complementary to the miRNAs of interest to fluorescent beads, whereby each bead represented a single miRNA. They added the beads to miRNAs isolated from cancer cell lysates and stained with streptavidin-phycoerythrin. When the samples were run through a flow cytometer, the instrument simultaneously measured bead color (miRNA identity) and phycoerythrin intensity (miRNA abundance) to reveal a different miRNA expression profile for each sample. The profiles turned out to be an accurate depiction of the developmental lineage and differentiation states of the tumors. Flow cytometry has benefited from many improvements on the instrument side, as well as the development of fluorescent antibodies and probes. The imaging cytometers and multiplex bead assays extend even further the power of flow cytometry by providing more information for a wider range of samples. Armed with the right software tools to analyze the data, flow cytometry is poised to pro vide an unprecedented close-up view of different cell populations and their molecular components, one cell (or one bead) at a time^ref
Web resources :
• Clinical Cytometry Society
• Compendium of Cytometry Internet Sites
• Purdue University Cytometry Laboratories (PUCL)
• Cytometry Software
• Cytometry Techniques and Protocols
• Median Fluorescence Intensity (MFI) Home Page by Eric Martz, University of Massachusetts
• Faculty of Medicine Flow Cytometry and Cell Sorter Facility
• Flow cytometry Home Page at the John Curtin School of Medical Research
• International Society for Analytical Cytology (ISAC)
• The Scripps Research Institute Flow Cytometry (TSRIFC)
• UF-ICBR Flow Cytometry Core Lab Home Page
• Immunofluorescent staining of intracellular cytokines for flow cytometric analysis
• Yeast cell cycle by flow cytometry by Susan Forsburg
• Procedure for setting compensation for multi-color flow cytometric analysis
• companies offering flow cytometry products and services : Abcam; Aber Instruments; Applied Cytometry Systems; Amersham Biosciences (GE Healthcare); Alternative Biomedical Services; Amnis Corporation; Anomeric Inc.; Applied Cytometry Systems;

Applied Precision, LLC; Bangs Laboratories; Beckman Coulter; BD Biosciences; Bioarray Solutions; Biocytex; Biodesign International; Biodetect; BioGenex; BioLegend; Biomeda; Bio-Rad; BioSource International; Biostatus Limited; BioSure; Bio-Tek; Caltag; Cell Sciences; Chromaprobe; Chromocell; Clontech (BD Biosciences); CompuCyte; Cytek Inc.; CytoBuoy; Cytometry Research, LLC;
DakoCytomation; De Novo Software; Diatec; Duke Scientific; eBioscience; Exalpha Biologicals Inc.; Fast systems; FlowCyte Associates; Gene Logic; Guava Technologies; Innovative Cell Technologies; IQ Products; Jackson ImmunoResearch Labs; Leinco Technologies; Linco Research Inc.; Luminex; Marligen Biosciences; Martek Biosciences Corp.; Media Cybernetics; Microbionix GmbH; MiraiBio; Molecular Probes (Invitrogen); Nikon; NPE Systems Inc.; Olympus; One Lambda Inc.; One Cell Systems; Orpegen Pharma;
Panbio Diagnostics; Partec; Perkin-Elmer; PPD Biomarker Discovery Sciences; Pharmingen (BD Biosciences); Phoenix Flow Systems;
Polysciences; Q-Biogene Inc.; Qiagen; R&D Systems; Research Diagnostics; Santa Cruz Biotechnology; Seradyn; Serotec; Sigma-Aldrich; Softflow; Southern Biotech; Spectrum Computing Services; Spherotech; Stem Cell Technologies; Surromed; Synthegen;
Sysmex; Tip labtech; Trevigen, Inc.; Tree Star, Inc.; Union Biometrica; Upstate; VayTek, Inc.; Vector Laboratories; Verity Software House; Zeiss
• isolation of WBCs and platelets from whole blood : hemolysis of RBCs
• isolation of lymphocytes, monocytes and platelets from whole blood
• Ficoll-Hypaque density gradient : overlay whole blood collected with an anti-coagulant on a Ficoll-Hypaque cushion (1:1) in a test tube. The Ficoll-Hypaque (Histopaque^®) is a polysaccharide (type II T-independent Ags (TI-2)) with a density (1.077 g/em³) higher than the density of lymphocytes and monocytes, but lower than the density of RBCs or granulocytes. Following centrifugation (30' at 2,500-2,700 rpm) the pattern in the test tube will be :
• plasma layer
• lymphocytes, monocytes, and platelets layer : they can be recovered by micropipetting and then washed  from Ficoll-Hypaque with serial dilution in complete RPMI medium and adjusted to a volume of 10 × 10⁶ cells/mL
• Ficoll-Hypaque
• RBCs and granulocytes will centrifuge to the bottom of the test tube
• removal of platelets by a single sucrose solution with a density of 65%
• incubation of whole blood with ADP and carbonyl iron prior to layering on a double discontinuous gradient of Ficoll-Hypaque selectively reduced the number of platelets and monocytes found at the upper lymphocyte interface after centrifugation. This modification in combination with a double discontinuous gradient results in a lymphocytes isolation method that is simple, rapid and reproducible^ref.
• isolation of lymphocytes from whole blood
• Polymorphprep™
• Nycodenz^®
• OptiPrep™
• Lymphoprep™(source : Axis-Shield) is a ready made, sterile and endotoxin tested solution for the isolation of pure lymphocyte suspensions. The solution contains sodium diatrizoate and polysaccharide in the following concentrations:
• sodium diatrizoate 9.1% (w/v)
• polysaccharide (Ficoll^®) 5.7% (w/v)
Physical-chemical characteristics:
• density 1.077 ± 0.001 g/ml
• osmolality 280 ± 15 mOsm
Principle of the separation technique : the most common technique for separating leucocytes is to mix blood with a compound which aggregates the erythrocytes, thereby increasing their sedimentation rate. The sedimentation of leucocytes is only slightly affected and can be collected from the upper part of the tube when the erythrocytes have settled. Using a mixture of sodium metrizoate and polysaccharide, Bøyum^ref (Bøyum, A?1968?: Separation of leucocytes from blood and bone marrow. Scand. J. Clin Lab. Invest, 21, Suppl. 97) developed a one-step centrifugal technique for isolation of lymphocytes. Thorsby and Bratlie^ref (1970) used this technique with only slight modifications in the preparation of pure lymphocyte suspension for cytotoxicity tests and lymphocyte cultures. As emphasized also by other authors, Harris and Ukayiofo^ref (1969), Ting and Morris^ref (1971) this is a reliable, simple and quick method suitable for the preparation of lymphocyte preparations from cadaver blood, and from anticoagulated blood stored at room temperature for up to 6 hours.
• Boyum A. Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol 1976;(Suppl 5):9-15.
Stability and storage : Lymphoprep™ is stable for 5 years provided the solution is kept sterile and protected from light. Prolonged exposure to direct sunlight leads to release of iodinine from the sodium diatrizoate molecule. This effect is negligible when working with this solution on a
day to day basis. Lymphoprep™ should be stored at room temperature.
Separation procedure :
• 1. Collect blood into a tube containing anticoagulant (EDTA, heparin, ACD) or use defibrinated blood.
• 2. Dilute the blood by addition of an equal volume of 0.9% NaCl.
• 3. Carefully layer 6 ml of the diluted blood over 3 ml Lymphoprep™ in a 12–15 mm centrifuge tube. Alternatively Lymphoprep™ can be underlayered. Avoid mixing of blood and separation fluid. Cap the tube to prevent the formation of aerosols.
• 4. Centrifuge at 800 x g for 20 minutes at room temperature (approximately 20°C) in a swing-out rotor. If the blood is stored for more than 2 hours, increase the centrifugation time to 30 minutes.
• 5. After centrifugation the mononuclear cells form a distinct band at the sample/medium interface, as shown in the figure. The cells are best removed from the interface using a Pasteur pipette without removing the upper layer.
• 6. Dilute the harvested fraction with 0.9% NaCl or medium to reduce the density of the solution and pellet the cells by centrifugation for 10 minutes at 250 x g.
Purity and viability : the described method has been found to be rapid, simple and reliable and gives excellent results with blood samples from most normal individuals and patients. The technique can also be used for preparation of lymphocyte suspensions for mixed lymphocyte culture tests. The contamination in the lymphocyte suspensions of erythrocytes is usually between 1-5% of the total cell number. Some immature granulocytes may follow the lymphocytes during intense immunosuppressive therapy. When heparinized blood is used, it is essential to remove most of the platelets, in order to avoid inhibition in the cytotoxicity test. The described washing procedure is usually sufficient.
• Biochemical testings
• fermentation tests detect fermented carbohydrate(s) and end products of fermentation(s). As the Bacteria excrete large amounts of lactic acid, acetic acid and formic acid, one can detect fermentation by using a differential medium containing a pH indicator dye.
• Neisseria enzyme test (NET) is a biochemical test that will differentiate between Neisseria gonorrhoeae, N. lactamica, Neisseria meningitidis, and Moraxella catarrhalis. A change of the original purple color to yellow will indicate N. meningitidis. A blue color will indicate N. lactamica. A pink/red color resulting from the addition of PRO reagent will indicate N. gonorrhoeae. If there is no color change, M. catarrhalis is indicated. Optimal incubation for this test is at a temperature of 37° C for a duration of 0.5 hours in ambient air.
• assays for specific enzymes secreted into the medium can be detected as for cell-free biochemical detections
• Pharmacology

Web resources :
• Apoptosis inducers
• Apoptosis inhibitors
• Cell synchronization reagents
• Guide to the selection of tetrapeptide caspase substrates
• Protein kinase A activators : cAMP analogs
• Protein kinase C subtypes
• Protein kinase G activators : cGMP analogs
• Specificities of selected Ser/Thr kinase inhibitors
• Selectivity of Tyr protein kinase inhibitors
• Selectivity of protein phosphatase inhibitors
• Selectivity of phosphodiesterase inhibitors
• Clonality assays :
• X-chromosome inactivation pattern (XCIP) / lyonization (the random inactivation of the X-chromosome in females that occurs early in embryogenic development^ref) : genes located on the X-chromosome and subject to X chromosome inactivation can be used to distinguish monoclonal from polyclonal cell populations in females. A prerequisite for all methods of clonality analysis based on X-inactivation is the ability to distinguish the paternally derived X-chromosome from the maternally derived one, based on common polymorphisms that exist in the general population at different loci on the X-chromosome^ref. A second prerequisite is the ability to distinguish the active from the inactive X-chromosome. Excessive skewing or clonality of hematopoiesis is defined as :
• corrected allele ratio (CR) > 3:1, which corresponds to the finding of one allele present on the same X-inactive chromosome in > 75% of cells^ref. The allele ratio is defined as the ratio between the intensity of PCR products from the 2 HUMARA alleles in a given sample, after digestion with methylation-sensitive enzymes (a'/a'b', a' being the smaller allele after digestion). The corrected ratio (CR) is defined as the allele ratio of the precut DNA sample (a'/a'b') divided by the allele ratio of the nonprecut sample (a/ab) specimen. This ratio compensates for potential preferential amplification of one of the two alleles (in general, the smaller one).
• Asimakopoulos equation : percentage of clonal granulocytes (Gc) was calculated using the following equation^ref, when Rt (T-cell ratio) > Rg (granulocyte ratio); Gc: t-Rg / Rt(Rg+1) × 100; when Rt < Rg Gc: Rg-Rt / Rg+1 × 100. This equation divides XCIP into 3 categories:
• clonal, when Gc>50% (corresponds to a Rg <0.33 in the presence of an Rt: 1.0)
• polyclonal, when Gc<50% and Rt>0.33
• ambiguous, when Gc<50% and Rt<0.33
Expression of the lower allele after correcting for the degree of lyonization in T-lymphocytes.
One potential limitation of X-inactivation assays is skewed X-inactivation pattern (XIP), which can mimic clonal derivation of cells
• costitutional excessive lyonization : females who have randomly inactivated a preponderance of one X-chromosome (either the paternal X or the maternal X) relative to the other, probably due to the stem cell pool size at the time of Lyonization
• acquired skewed XIP skewed XIP has been shown to increase with age, with > 30% of the normal population having skewed XIP at 60 years^ref. Gale et al^ref found significant skewed XIP in 23% of normal females using PGK and HPRT probes
21–31% of healthy women have a skewing pattern of X-inactivation (or lyonization) with techniques based on DNA polymorphism^{ref1, ref2}. Different hypotheses have been suggested to explain the especially high rate of excessive lyonization in hematopoietic tissue :
• skewing of the X-chromosome inactivation pattern may be caused by the small stem-cell pool size at the time of X-chromosome inactivation^ref. Patterns will therefore vary from tissue to tissue. Because X-chromosome inactivation takes place gradually over a period of time, as demonstrated by Tan et al., this will influence the patterns obtained; e.g., inactivation of the progenitor blood cell pool at an earlier development time would lead to considerably more skewing in blood cells than in gut cells, which are apparently one of the last cell populations to be inactivated^ref. Second, it is possible that somatic cell selection occurs after random X-inactivation has been established and as a consequence of a selective pressure on blood cells^{ref1, ref2, ref3}. Finally, stem-cell depletion or the development of clonal hematopoiesis may also induce a nonrandom pattern of X-inactivation^ref. Each of these acquired processes could result in highly skewed X-inactivation ratios and should be associated with an increased incidence of skewing with age. Fey et al. have reported data supporting a different incidence in skewing between elderly and younger females^ref, although Gale et al. were unable to demonstrate an effect of age on skewing when the totality of published data for healthy females was considered (n = 100)^ref. Recently, Busque et al. showed that incidence of excessive lyonization in healthy women increased significantly with age with the HUMARA and PGK genes. The incidence of skewing (allele ratios > 3:1) was 8.6% (14 of 162) in neonates, 16.4% (11 of 67) in women 28 to 32 years old, and 37.9% (25 of 66) in women 60 years^ref. A statistically significant difference in skewing rate in neonates using DNA polymorphism (24%) compared with RNA polymorphisms (3%) has been reported. One explanation could be that methylation was incomplete at birth on one allele, paternal or maternal. Indeed, the opposite pattern was observed in one elderly woman and two ET patients. In the case of the elderly woman, the discrepancy could be due to hypermethylation of DNA on the active X-chromosome resulting in incomplete digestion and consequently a random pattern, while basically X-inactivation was skewed. Alternatively, the occurrence of a mutation on one allele of the IDS gene may lead to the absence of its expression in females with a random pattern of X-inactivation. In patients, it may be due to hypermethylation of DNA as already described in tumors^ref, especially because of the clear clonality feature obtained by transcript analysis in one patient (clonality in granulocytes and platelets with polyclonality in T lymphocytes). Comparison between the DNA and RNA polymorphisms showed some interesting results that may help clarify the interpretation of excessive lyonization in healthy females. Both techniques showed an increase in skewing rate between females <50 years and elderly ones. This suggests that methylation does not increase with age, but that depletion of stem cells, clonal selection, or the appearance of clonal hematopoiesis might occur with age. Furthermore, using RNA polymorphisms, we confirmed the findings of others that incidence of skewed lyonization is very low at birth. This low incidence of excessive skewing in neonates also suggests that the estimated number of stem cells present at the time of X-inactivation could be higher than previously reported. These results are not in agreement with those of other authors who found a low rate of nonrandom X-inactivation at birth using HUMARA polymorphism^ref, but the latter group of neonates was much smaller (40 vs 162)^ref
• technical reasons may account for some of the discrepancies in the estimation of skewed lyonization in healthy females and possibly clonality analysis in various malignancies. This hypothesis is supported by the low frequency of skewed lyonization found by studying the expression of G6PD polymorphism at the protein level compared with the high frequency found with methylation analysis of the M27ß locus^{ref1, ref2, ref3}
Skewed XIP of polyclonal cells can only be distinguished from clonal cells using appropriate tissue controls. Because XCIP may vary from tissue to tissue^ref, somatic control from embryologically related tissue (such as T lymphocytes or buccal mucosa cells) is needed to interpret skewed patterns of X-inactivation in BM cells^ref. For myeloid disorders in the young, T lymphocytes serve as a suitable control XCIP, but interpretation of imbalanced XCIPs in the elderly can be difficult^ref. T lymphocytes are thought to be the most suitable control for disorders of myeloid cell origin, because they originate from the same pluripotent stem cell, and in younger hematologically normal individuals, they have the same XCIP^ref. However, studies have also shown that there is an increased incidence of skewing in myeloid cells of the elderly^{ref1, ref2}, which may not be reflected in T-lymphocyte XCIPs^{ref1, ref2}. In a study, for example, an extremely skewed pattern with > 90% expression of one allele was found in the neutrophils of 33% and T lymphocytes of 9% of hematologically normal females more than 75 years of age, but in only 3% of younger individuals^ref. In the few studies of ET patients in which T-lymphocyte XCIPs have been compared with neutrophil patterns, the results have been conflicting. Tsukamoto et al^ref showed monoclonal XCIPs in neutrophils of all 13 patients in their study, suggesting that ET is almost invariably a clonal disorder, although we would only consider 6 of these patients to be assessable because of advanced age or constitutive skewing of the T cells. In a study of 46 patients by El-Kassar et al^ref, the majority seemed to have monoclonal neutrophils (61%), but 14 patients had polyclonal patterns in both neutrophils and T lymphocytes. Ten of these 14 patients could be studied using RNA polymorphisms. Platelet polyclonality was confirmed in 7 patients, and monoclonal platelets were found in 3 patients, these latter results suggesting that ET could sometimes be restricted to the megakaryocytic line. Such lineage restriction is in marked contrast to other clonal myeloproliferative disorders^ref.
• DNA methylation patterns-based clonality assays (MCA) on active and inactive X-chromosome alleles. It uses Southern blotting or PCR of either ... :
• restriction enzyme polymorphisms, eg :
• HPRT-PCR clonality assay
• phosphoglycerate kinase (PGK)-PCR clonality assay (PPCA) : active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanine-plus-cytosine-rich island at the 5' end of the gene^ref
• variable number tandem repeat (VNTR) sequences, eg :
• M27b, a highly informative probe detecting a polymorphic X chromosomal locus, DXS255. The polymorphism detected at this locus is due to variable numbers of tandem repeats. The rate of constitutional heterozygosity detected by M27b was 88%. Normal tissue from gastrointestinal mucosa and thyroid showed random, hence polyclonal, patterns. Nonrandom clonal X inactivation was detected in all 22 malignant neoplasms that had been shown to be clonal by other DNA markers, such as antigen receptor gene rearrangements or clonal loss of heterozygosity at 17p and other loci. 16/48 normal blood leukocyte samples (33%) showed considerably skewed X inactivation patterns. Comparison of blood leukocytes and normal tissue indicated that in a given individual, X inactivation patterns may be tissue specific. M27b was used to study the clonal composition of 13 benign thyroid nodules from 12 multinodular goiters with rapid recent growth, traditionally termed "adenomas." 9 of them were clonal, whereas 4 nodules and tissue from a case of Graves' goiter were not, indicating that some, but not all, such thyroid nodules may represent true clonal neoplasms. The M27b probe permits one to study the clonal composition by the X inactivation approach of a wide variety of solid tumors from most female patients. As a control, normal tissue homologous to the tumor type of interest is preferable to DNA from blood leukocytes, since the latter may show nonrandom X inactivation patterns in a fairly high proportion of cases. M27b may, therefore, be of limited use for the clonal analysis of neoplasms derived from hematopoietic cells^{ref1, ref2, ref3}
• human androgen receptor assay (HUMARA)^ref. It offers several advantages over other X-inactivation-based assays because it has a high rate of heterozygosity (>90%) as a consequence of > 20 alleles generated by the different number of trinucleotide (CAG) repeats in the first exon at this locus in the general population^ref. The close proximity of the polymorphic trinucleotide repeat to 4 methylation sites^ref permits a clonal analysis based on the PCR^ref. One primer is labeled at the 5' end with fluorescein. The products were analyzed and quantified with an automated DNA sequencer (ALF, Pharmacia-Biotech). Under optimal conditions amplification efficiency of two HUMARA alleles is near-equivalent, generating PCR products in a ratio proportional to that of the genomic template. In contrast, reduction of template quantity, damage of template by ultraviolet irradiation or addition of monovalent salts (sodium chloride, sodium acetate or ammonium acetate) produces highly variable imbalances of allelic PCR products, with a strong tendency to preferentially amplify lower molecular weight alleles. Variability and biasing was diminished by substitution of 7-deaza-2'-dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and intermolecular GC base pairing^ref. This assay has been validated in the analysis of hematopoiesis and in various hematologic disorders^{ref1, ref2, ref3, ref4} (Busque L, Maragh M, DeHart D, McGarigle C, Vose J, Armitage J, Meisinger D, Wheeler C, Gaines L, Belanger R, Habel F, Dunbar C, Champagne M, Gross W, Weinstein H, Antin JH, Gilliland DG: Clonality of bone marrow repopulation after allogeneic and autologous bone marrow transplantation (ABMT). Blood 82:457a, 1993; Busque L, Gilliland DG: Clonality analysis in myelopoietic disorders. Focus Growth Factors 5:3, 1994).
• transcription-based clonality assays (TCAs) to detect mRNA polymorphism^ref : only genes on the X-active chromosome are transcribed^{ref1, ref2, ref3}. One advantage of this technique is the possibility to study clonality in nonnucleated cells such as platelets and reticulocytes, which lack DNA. Moreover, it avoids the problem of potential incomplete digestion of genomic DNA^ref
• HUMARA : positive^ref and negative reports^ref
• glucose-6-phosphate dehydrogenase (G6PD) : only 18% of females in the USA are informative (heterozygous) for this chromosome with a test based on nucleotide #1311 exonic polymorphism. Detected using rtPCR-LDR
• p55 / MPP1 : a palmitoylated membrane protein with the G or T at cDNA #358 at the third exon (38% of Caucasian, African-American, and Asian females are informative)^ref. Detected using rtPCR-LDR
• iduronate-2-sulfatase (IDS) : 41% were informative^ref; the frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; not informative in Oriental females^ref
• 4-and-a-half LIM domain 1 (FHL1)^ref
• Bruton tyrosine kinase (BTK)^ref
• combinations :
• G6PD + p55 : there is a multiple crossover between the G6PD and the p55 polymorphisms (separated by approximately 200 kb), suggesting that these two polymorphisms are in linkage disequilibrium; thus, approximately 50% of female subjects are informative for clonality studies using the 2 assays^ref. Allele-specific PCR (ASPCR) is a simple, reproducible assay, practical for screening genomic DNA samples for p55/G6PD genotypes, rapid clonality determination, and to determine the linkage relationship between these closely related loci. The salient feature of ASPCR is the performance of two PCR rounds. The first generates template; the second, using one aliquot of first-round products in two reaction tubes, each containing one allele-specific primer, detects each allele. ASPCR and rtPCR-LDR produced identical p55/G6PD results in 91 normal female genomic DNAs, and in 12 clonal hematopoietic disorder cDNAs, confirming assay validity : 60% of females were heterozygous at one or both loci. G6PD and p55 allelic frequencies were significantly different among African-American men and women, but were not significantly different among Caucasian men and women. These loci were in linkage equilibrium among African Americans, but not among Caucasians^ref.
• G6PD + p55 / MPP1 + IDS : 76% of Caucasian females and 62% of African-American females are informative for these assays^ref
• G6PD + IDS + p55 / MPP1 establishes the presence or absence of clonal hematopoiesis in about 65% of female subjects
• G6PD + IDS + p55 / MPP1 + BTK + FHL1 establishes the presence or absence of clonal hematopoiesis in about 90% of female subjects^ref
• protein polymorphism :
• G6PD isoenzymes^ref (Fialkow^{ref1, ref2, ref3}) : limited by the low frequency of heterozygotes
Clonality assays involving DNA and RNA polymorphisms are usually concordant except in neonates^ref
• myeloid-specific clonality assays :
• JAK2(V617F) in polycythemia vera (PV) > other classic and atypical myeloproliferative disorders (MPD)^ref
• BCR-ABL in chronic myeloid leukemia
• PML-RARa
• FIP1L1-PDGFRA
• CBFb/MYH11
• AML1/ETO
• FLT-3 mutations
• other tranlocations
• lymphoid-specific clonality assays :
• in B-cell lymphoproliferative disorders : BCR
• PCR using a consesus primer paired to FR3 in V_H region and another paired to J_H region, so amplifying the D_H region
• in T-cell lymphoproliferative disorders : TCR
• PCR analysis of length of CDR3 of the rearranged TCR gene, followed by sequencing
• bands other than the germ-line bands on Southern blots probed for the TcR b gene (Vb)
• anti-TCRVb monoclonal antibody staining
• clonogenicity assays :

Indications :
• analysis of carcinogenic transformation
• effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells
• studies of stem/progenitor cells
The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. Analysis of the features of cell colonies by laser scanning cytometry (LSC) provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies^ref.
• Nucleic acid extraction
• manual extraction
• automated extraction : multilayer soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels containing on-off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based devices. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained^ref.
• from a group of cells : a series of tiny O-shaped channels, each of which serves as a mixing vessel, is carved in a rubber postage stamp-sized chip that drags DNA from cells. A network of inlet canals and valves feeds each O-channel with a mixture of cells and the chemical solutions that dilute and burst them. Tiny moving plugs in each O-channel swirl the contents like a cement mixer, so that the cells pop and release their DNA, which is sucked out the other side
• from a single cell : for example, to identify a rare cancerous blood cell that is about to trigger a leukaemia relapse but is swimming among healthy neighbours. Most techniques today tend to analyse groups of cells, which can mask a single cell's erratic behaviour. Some researchers have started analysing single cells with conventional laboratory technique, but shrinking the size of each chemical reaction cuts the cost of reagents and slashes the time taken for each step
• isolation of serum from the red blood cells from whole blood :
• draw a volume of whole blood into a Vacutainer without using any anti-clotting factors.
• allow blood to clot at 37°C for 30 to 60 minutes.
• pull the rubber stopper from the Vacutainer tubes and using a long Pasteur pipette separate the clot from the edges of the inside of the glass.
• set the tubes at 4°C overnight to allow the clot to contract.
• decant the serum from the clot into a centrifuge tube (don't worry if some red blood cells and clot material decant along with the serum).
• centrifuge at 3,000 X g for 10 min to remove remaining red blood cells and clot material.
• decant the supernatant (serum) and aliquot in labeled tubes for storage.
• if azide won't interfere with your applications, you should add 5-10 mM sodium azide to this working solution tookeep bacteria from growing in it.
• there are various methods of storing prepared serum. If the serum is a working solution one can store it at 4°C. To keep bacteria from growing in the solution you can add sodium azide (5-10 mM final concentration) but only if the sodium azide won't interfere with other uses of the serum. Alternatively, serum can be stored at -80°C. Antibody will be stable for months if the serum solution is stored at -80°C.
• Imaging
• following protein dynamics in living cells
• photoactivation techniques work by converting molecules to a fluorescent state by using a brief pulse of high-intensity irradiation
• flash photolysis of caged compounds : caged fluorophores are fluorophores that have been chemically modified with a caging group that quenches their fluorescenc until a brief pulse (usually < 100 ms) of 350-nm light breaks the photolabile bond connecting the fluorophore and the caging group
• photobleaching techniques : the photo-induced alteration of a fluorophore that extinguishes its fluorescence.
• fluorescence recovery after photobleaching (FRAP) : a region of interest (ROI) is selectively photobleached with a high-intensity laser and the recovery that occurs as molecules move into the bleached region is monitored over time with low-intensity laser light. Depending on the protein studied, fluorescence recovery can result from protein diffusion, binding/dissociation or transport processes. Analysis of fluorescence recovery can be used to determine the kinetic parameters of a protein, including its diffusion constant, mobile fraction, transport rate or binding/dissociation rate from other proteins. In experiments in which the protein of interest moves freely, the fluorescence will recover to the initial prebleach value and the shape of the recover can be described mathematically with a single component recovery. Determining the effective diffusion coefficient (D_eff) and mobile fraction (M_f) of a protein from such data is relatively straightforward, given the previous analysis of FRAP kinetics. If the shape of the curve is complex (that is, it requires a multi-component diffusion equation), then multiple populations of the molecule with differing diffusion rates are present. This can occur when a molecule undergoes binding and release from intracellular components or exists as a monomer and multimeric forms. Alternatively, the protein might not be diffusing but might be undergoing movement driven by molecular motors or membrane tension flow. A simple test for determining whether a fluorescent protein moves by diffusive movement or facilitated transport is to vary the size of the bleached area or beam radius, w. The recovery will change with an w² dependece for diffusive movement only. Accurate analysis of FRAP data requires that the bleach event is much shorter than the recovery time and preferably as short as possible. Moreover, the recovery event must be monitored until a recovery plateau is achieved, which is much greater than the half-time for recovery. Images on the confocal microscope are obtained by scanning a focused laser beam across the specimen and recording the emitted fluorescence through a pinhole that is situated in front of the light detector. One way to photobleach using this system is to define a region-of-interest at the highest possible zoom, set the laser power to maximum, and set the laser attenuation to zero. The high zoom increases the dwell time of the laser on the bleached region per line scan (laser intensity increases proportionally to the square of the zoom factor), which therefore greatly increases the radiation per area. But a more advanced method is to use an acousto-optical tunable filter (AOTF; available on more recent commercially available confocal microscopes), which allows rapid (microsecond to millisecond) attenuation of the laser as it scans as field. By allowing rapid switching between the bleaching and normal beam, the AOTF allows accurate measurements of diffusion rates in defined areas. Use of an AOTF also enables users to photobleach virtually any pattern or shape. This allows FRAP studies to be done on organelles of complex shapes, allowing the lateral mobility of organelle-specific membrane and lumenal proteins to be investigated. Selective photobleaching on a confocal microscope also provides a method for analysing aspects of protein dynamics other than diffusion (incuding assembly/disassembly of protein complexes in cells), the exchange of cytosolic proteins on and off organelles, and the lifetime and fate of membrane-bound transport intermediate. This type of analysis often requires measuring the fluorescence signal of GFP in a specific structure or area, to compare it with fluorescent molecules in different sites of states are known, computer modelling can then be used to determine the parameter values (that is, the rate constants for binding interactions and exchange times) of the processes of interest.

Problems :
• lack of recovery or partial recovery after photobleaching : possible explanations include :
• an immobile fraction of unbleached molecules in the cytoplasm that could not diffuse into the bleached region
• an immobile fraction of molecules in the bleached area that was unable to exchange with the incoming unbleached molecules
• the bleached area is not continuous with the rest of the cell (for example, a separate membrane compartment)
• reversible photobleaching of GFP : the excitationmight cause the GFP molecule to flicker or to be sequestered in a triplet state. Both of these situations can result in the recovery of fluorescence (in miliseconds to several seconds) of the GFP molecule in the absence of diffusion. To control for any reversible photobleaching of the GFP in a FRAP experiment, the FRAP conditions should be repeated in fixed samples in which no recovery of fluorescence should be expected. Or, alternatively, the bleach spot size could be varied and the changes in the timescale of recovery could be confirmed
• non-diffusive behaviour : measurements in FRAP studies are often complicated by the binding and dissociation of fluorescent molecules to and from intracellular components. This is usually reflected in the FRAP curves by the longer recovery times, by an incomplete recovery (an immobile fraction) or by the presence of several slopes (indicating several recovery processes over different timescales). Kinetic modelling methods, along with computer simulations, have been useful tools to dissect and analyse the recovery curves obtained by FRAP. A kinetic model is characterized by biophysical parameters, such as binding and release rate constants, diffusion constants, flow rates and residence times. The model can be simulated on the computer for different parameter values. Once the parameters that best fit the experimental data have been determined, the predictions of the model can be tested experimentally
• D values of the same region of interest (ROI) in the samne cell in 2 consecutive experiments are different : a potential explanation is damage to the photobleached area. Decreasing the bleach time, acquisition time or the excitation beam intensity during the recovery period could avoid damaging the cell. Using YFP rather GFP or CFP will also make it easier to photobleach
• strategies that indirectly highlight a pool of molecules by decreasing the background fluorescence.
• inverse FRAP (iFRAP) is performed as a normal FRAP experiment with the exception that the molecules outside a region of interest are photobleached and the loss of fluorescence from the non-photobleached region is monitored over time. As opposed to the rate of recovery studied using a FRAP experiment, iFRAP offers a way to monitor the rate of movement out of a region. This method
• fluorescence localization after photobleaching (FLAP) : the same protein-of-interest is tagged with 2 different fluorophores that co-localize when expressed in cells. By photobleaching one of these fluorophores, a selected pool can be highlighted and followed over time.
• fluorescence loss in photobleaching (FLIP) : a fluorescent cell is repeatedly photobleached within a small region while the whole cell is repeatedly imaged. Any regions of the cell that are connected to the area being bleached will gradually lose fluorescence due to  lateral movement of mobile proteins into this area. By contrast, the fluorescence in unconnected regions will not be affected. In addition to assessing continuity between areas of the cell, FLIP can be used to assess whether a protein moves uniformly across a particular cel compartment or undergoes interactions that impede its motion. Furthermore, it can be used to reveal faint fluorescence in unconnected compartments that normally cannot be seen against the bright fluorescence that arises in other parts of the cell.
• donor FRAP (DFRAP)
• the optical voltage sensor FlaSh, made from a fusion of a GFP "reporter domain" and a voltage-gated Shaker K⁺ channel "detector domain," has been mutagenically tuned in both the GFP reporter and channel detector domains. The biarsenic compound FIAsH, a compound that fluoresces green when bound to a genetically engineered protein motif (TC motif). This has produced sensors with improved folding at 37°C, enabling use in mammalian preparations, and yielded variants with distinct spectra, kinetics, and voltage dependence, thus expanding the types of electrical signals that can be detected. The optical readout of FlaSh has also been expanded from single wavelength fluorescence intensity changes to dual wavelength measurements based on both voltage-dependent spectral shifts and changes in FRET. Different versions of FlaSh can now be chosen to optimize the detection of either action potentials or synaptic potentials, to follow high versus low rates of activity, and to best reflect electrical activity in cell types with distinct voltages of operation
Web resources : An introduction to chemiluminescence and bioluminiscence measurements
 
• laser nanosurgery
• With pulses of intense laser light 10^-15 s long, they can vaporize subcellular structures inside living cells without killing them. The laser works inside the cell without damaging the surface : the light is focused extremely tightly, using a microscope, into a space a few hundred nm across.
• transfections
• transfection of nucleic acids
• aims
• transfection of proteins
• transfection of every kind of molecule
• transfection ways
• atomic force microscope (AFM) can perform keyhole surgery, adding or removing molecules from precise locations inside a single living cell without harming it. Using microscopic lances to remove material from fertilized eggs is now a routine technique, but these microcapillaries are still quite clumsy and difficult to control precisely without damaging the cell : as they press through the cell wall, it is often deformed so badly that the cell dies. Unlike a microcapillary, the AFM can sense the force it exerts on the cell, making it extremely responsive. A beam of energetic ions is used to sharpen a standard silicon AFM tip into a needle just 8 mm long and 200 nm wide. When the researchers inserted the needle into a human embryonic kidney (HEK) cell, the cell wall was indented by only 1 mm, much more delicate than comparable microcapilliary procedures. The cell membrane quickly returned to its original shape, and the needle was pushed into the cell's nucleus. It would also be possible to monitor the chemistry of a cell in real time by coating the needle's tip with molecules that grab onto chemicals produced inside the cell. If the tip were designed to fluoresce when the molecules bind, or to change its electrical charge in a detectable way, scientists could watch the cell's response to a new drug. The tool could be particularly useful for studying human cells in diseases such as asthma or cystic fibrosis in which cells are often in short supply^ref
• phagocytosis of molecule-loaded autologous RBCs modified to be selectively recognized by tissue macrophages, e.g. by promoting autologous Ig binding and C3b deposition. However, several molecules have been shown to leak rapidly through RBC membrane due to simple diffusion. Other molecules may be toxic to the RBC itself, thus preventing their use as a carrier system. It is interesting to note that RBCs are "active" carrier systems, endowed with a number of enzimatic activities that can be conveniently explored to convert an inactive pro-drug into an active drug. This property permits the design of a number of pro-drugs that can be synthetised with charged chemical groups making them non-diffusible or non-toxic. Once these chemical groups have been hydrolyzed by resident RBC enzymes, the pro-drug is converted into an active drug that can diffuse through the RBC membrane and thus released in circulation or at specific sites when RBC targeting is achieved.
• artificial protocells are not truly alive, because they cannot replicate or evolve, but they can churn out proteins for days, and could be useful for drug production, as well as advancing the quest to build artificial life from scratch. Vincent Noireaux and Albert Libchaber of the Rockefeller University in New York have managed to package up some of the molecular machinery of a cell inside an artificial, bacterium-sized membrane. And they can perforate the membrane with holes that allow nutrients and energy-rich molecules to get into the cells from the surroundings. These protocells contain all the machinery needed to generate proteins from amino acids, so they could be used as miniature factories, to produce proteins of industrial and medical value, routinely produced by genetically engineered bacteria bred in fermentation vats. But artificial cells would make much simpler protein factories, perhaps more easily tailored to make specific products. Ready-made mixtures of all the biomolecules that a cell needs for protein production are commercially available, extracted from bacteria such as Escherichia coli. These mixtures can make specific proteins, but they stop working within about 2 hours unless they are continually fed with raw materials and cleaned of waste products. To enclose this biomolecule mixture inside membranes like those of natural cell walls, Noireaux and Libchaber made microscopic droplets of the cell extract suspended in oil. Soap-like molecules called phospholipids then coated the surface of these droplets in the same way that emulsifying agents surround the droplets in a salad dressing, stopping them from coalescing.The researchers then coated the droplets with a second layer of phospholipids, to form a double layer that looks just like the membrane of a real cell. To monitor the behaviour of their cells, Noireaux and Libchaber added DNA that encodes a fluorescent protein, so that as the cells produce it, they start to glow. Whereas the bare cell extract ran out of steam after 2 hours, wrapping the molecules in a membrane kept the system 'alive' for more than twice as long. To get raw materials into the protocells from the surroundings, the researchers added a bacterial gene that encodes a protein called alpha-hemolysin. This protein has a hollow, barrel-shaped structure and it inserts itself into cell membranes to create pores. Once fitted with these molecular portals, the cells kept churning out the fluorescent protein for days. Noireaux and Libchaber are now working on fixing molecules to their protocell walls that will pinch the membranes together so that the cells can divide, as bacteria do. This could be the first step in making artificial cells that replicate. Another big challenge is to enable the transcription and translation apparatus to make copies of itself, so that the new cells can both function like their 'parent'.

Web resources : Liposomes as models for biological cells - the minimal cell project
• tissue microarrays to discover not only the location and abundance of all human proteins, but also how these are affected by disease states. From each cloned gene from chromosomes X, Y, 14, and 22 (more will follow), the team produces polyhistidine-tagged recombinant protein and uses it to generate polyclonal antibodies. In formalin-fixed tissue samples on a slide, the proteins are denatured, unfolded. If you're using monoclonal antibodies, you're lucky if the one antibody-binding site is exposed. The group is producing 100–150 new recombinant proteins each week, and five new antibodies a day. They are generating about 150 gigabytes of data every day, and have written 60,000 lines of code in the past year to create a database of the images and associated experimental data. Each antibody is used to produce more than 700 pictures from different tissue samples, amounting to 10,000 images each week. The fully automated procedure probes tissue microarray samples from 48 normal tissues, including heart and brain, and from the 20 cancers that occur most frequently in the Western world, including breast, prostate, and liver.


Web resources : Swedish Human Proteome Resource (HPR)

Copyright © 2001-2005 Daniele Focosi. All rights reserved | Terms of use | Legal notices
 About this site  | Acknowledgements  |  Open forum  |  Tell a friend about this site  | Current link partners
Abbreviations and acronyms  |  Medical terminology  |  Add a link  | Translate this site into your favourite language  | Softwares

---