In vivo analysis techniques : molecular labeling

IN VIVO ANALYSIS TECHNIQUES : MOLECULAR LABELING

quantum dots (QDs) : a quantum dot is a potential well that confines electrons in 3Ds to a region of the order of the electrons' de Broglie wavelength in size, a few nanometers in a semiconductor. Compare to quantum wires and quantum wells. Because of the confinement, electrons in the QD have quantized, discrete energy levels, much like an atom. For this reason, QDs are sometimes called "artificial atoms." The energy levels can be controlled by changing the size and shape of the quantum dot, and the depth of the potential. In semiconductors, quantum dots are small regions of one material buried in another with a larger band gap. QDs occur accidentally in quantum well structures due to monolayer fluctuations in the well's thickness. Densely-packed QDs form spontaneously under certain conditions during molecular beam epitaxy when a material is grown on a substrate to which it is not lattice matched. The resulting strain results in grown in pyramid-shaped QDs. Individual QDs can be created by a technique called electron beam lithography, in which a pattern is etched onto a semiconductor chip, and conducting metal is then deposited onto the pattern. Being quasi-zero dimensional, QDs have a sharper density of states than higher-dimensional structures. As a result, they have superior transport and optical properties, and are being researched for use in diode lasers and detectors. QDs are one of the most hopeful candidates for quantum computation. By applying small voltages to your leads, you can control the flow of electrons through the QD and thereby make precise measurements of the spin and other properties of the quantum dot. With several entangled QDs (qubits), plus a way of performing operations, quantum calculations might be possible.

monitoring interactions within and among cells as they grow and differentiate is a key to understanding organismal development. Fluorescence microscopy is among the most widely used approaches for high-resolution, noninvasive imaging of live organisms^ref1,
ref2, and organic fluorophores are the most commonly used tags for fluorescence-based imaging. Despite their considerable advantages in live cell imaging, organic fluorophores are subject to certain limitations. Fluorescent QDs are inorganic fluorescent nanocrystals that overcome many of these limitations and provide a useful alternative for studies that require long-term and multicolor imaging of cellular and molecular interactions^ref1,
ref2. For labeling specific cellular proteins, QDs must be conjugated to biomolecules that provide binding specificity. Bioconjugation approaches vary with the surface properties of the hydrophilic QD used. The mixed surface self-assembly approach is recommended for conjugating biomolecules to QDs capped with negatively charged dihydroxylipoic acid (DHLA). In this approach DHLA-capped QDs are conjugated to proteins using positively charged adaptors^{ref1,
ref2,
ref3}, for example, a naturally charged protein (e.g., avidin^ref), a protein fused to positively charged leucine zipper peptide (zb)^ref or a protein fused to pentahistidine peptide (5 His)^ref. The use of avidin permits stable conjugation of the QDs to ligands, antibodies or other molecules that can be biotinylated, whereas the use of proteins fused to a positively charged peptide or oligohistidine peptide obviates the need for biotinylating the target molecule. This procedure describes the bioconjugation of QDs and specific labeling of both intracellular and cell-surface proteins^ref1,
ref2.

reagents

Quantum dots (QDs; e.g., Quantum Dot Corporation or Evident Technology)
Cells or tissue for labeling, prepared appropriately depending on the application (QDs can be used to tag live cells, label cell-surface proteins, or label fixed cells or tissue sections)
Amylose resin (New England Biolabs)
Antibodies of interest
Avidin (Sigma Chemicals)
Bovine serum albumin (BSA), 1% in PBS
Lipid-based transfection reagent (e.g., Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche))
Maltose (Sigma Chemicals)
Maltose-binding protein fused to the basic leucine zipper domain (MBP-zb) and protein G fused to the basic leucine zipper domain (PG-zb) expressed and purified from bacteria as described elsewhere^ref.
Phosphate-buffered saline (PBS; Sigma Chemicals)
Sodium tetraborate (Sigma Chemicals)
Sulfo-NHS-SS biotin (Pierce Biotechnology)
Tris-buffered saline (TBS; Sigma Chemicals)

equipment

Hand-held UV lamp
Fluorescence microscope (for details, see the section Imaging the Labeled Cells)

bioconjugation of the QDs

1. Prepare the QD mix: combine 200 pmol avidin and 600 pmol MBP-zb; to this mixture add 100 pmol QD and bring the final volume to 200 ml with 10 mM sodium tetraborate buffer (pH 9.0). QDs are often synthesized and conjugated to specific biomolecules in the investigator's laboratory. We use CdSe-ZnS QDs, which are rendered water soluble by capping with DHLA as described elsewhere^ref. Quantum Dot Corporation provides QDs conjugated to avidin for use with biotinylated proteins and antibodies. Evident Technology offers biotin-conjugated QDs and QDs that can be conjugated to the N or C terminus of a protein. Both suppliers provide QDs conjugated to specific antibodies as well as protocols for conjugating proteins to their QDs. To regulate the number of linker molecules (e.g., avidin) on each QD, the molar ratio of MBP-zb to the linker molecule should be altered. Because of their net positive charge, these proteins compete with each other to bind the negatively charged DHLA coat on the surface of the QD. Altering the ratio of these proteins in the mixture facilitates regulating their relative numbers on each QD. Because these proteins bind to QDs in a competitive manner, it is critical that both proteins be mixed thoroughly before addition of QDs. Nonhomogeneous mixing could result in greater variation in the ratio of the two molecules on each QD. The ratio presented here results in an average of one avidin molecule present for 3 MBP-zb molecules bound to each QD. To optimize the number of linker molecules, a series of QD bioconjugates should be prepared with ratio of linker to MBP-zb above and below the suggested ratio of 1:3. These ratios should be individually tested for specificity and affinity of binding before deciding upon the best QD bioconjugate for cellular labeling.

2. Allow the mixture to stand at 20–25 °C for 15 min.
3. Add an additional 150 pmol MBP-zb to the mixture and let it react at 20–25 °C for a further 15 min.
4. Set up a 500 ml amylose column, equilibrated with 10 mM sodium tetraborate buffer.
5. Load the entire preparation of the QD bioconjugate mix (from step 3) onto the column and wash the column twice with sodium tetraborate buffer.
6. Add 200 pmol of the biotinylated molecule of interest (in the case of avidin-conjugated QDs) or specific antibody (in the case of specific antigen-conjugated QDs) to the column and let it react at 20–25 °C for 30–60 min.
7. Wash the column twice with sodium tetraborate buffer and elute using 10 mM maltose (in PBS or sodium tetraborate buffer) until all the QDs are eluted from the column (QD elution can be easily monitored by placing the column in UV light and monitoring the QD fluorescence of the eluant)^ref. This approach provides a pure population of conjugated QDs, free of unbound QDs and of the unbound biomolecules. If the QD bioconjugates do not elute from the column, use a fresh preparation of MBP-zb and if the problem persists try a fresh batch of QDs.
8. To label live cells, follow option A, Labeling of cell-surface proteins in live cells; for labeling fixed cells, proceed to option B, Labeling of proteins in fixed cells. Bioconjugated QDs are used for the specific labeling of both intracellular and cell-surface proteins^ref1,
ref2. In live cells, however, these approaches permit labeling only of the cell-surface proteins^ref.
Option A. Labeling of cell-surface proteins in live cells

1. Wash the cells with fresh growth medium.
2. Incubate the cells for 30 min, at either 37 °C or 4 °C, in growth medium containing the appropriate amount of QDs bioconjugated to biotinylated ligand or antibody (from step 7 above). Incubating the cells at 4° C will help to minimize endocytic uptake of ligands, antibodies and QD bioconjugates.
3. Remove excess QD bioconjugates by washing the cells two or three times with growth medium or PBS. When using biotinylated ligand or antibodies, continue with step 4.
4. Incubate the cells for 10–15 min (at 37 °C or 4 °C) with avidin-conjugated QDs.
5. Remove the excess unbound QDs by washing the cells two or three times with growth medium or PBS. These cells can be monitored live or subsequently fixed using chemical fixatives such as 4% paraformaldehyde without affecting the QD fluorescence^ref.
6. To visualize results, proceed to the section Imaging the Labeled Cells. If you find the QD bioconjugates cause aggregation of labeled proteins on the surface of live cells, use the mixed surface conjugation approach

Option B. Labeling of proteins in fixed cells

1. Fix the cells using appropriate chemical fixative and wash the cells 2-3 times with PBS.
2. Incubate the cells for 30 min at 20–25 °C in 1% BSA in PBS.
3. Replace the 1% BSA solution with an appropriate amount of QD bioconjugates or biotinylated antibody or ligand prepared in 1% BSA solution and incubate at 20–25 °C for 30–45 min.
4. Wash the cells two or three times with PBS.
When using biotinylated ligand or antibodies continue with step 5.
5. Incubate the cells for 10–15 min at 20–25 °C with QD avidin, and then wash two or three times with PBS to remove excess unbound QD avidin.
6. To visualize results, proceed to the section Imaging the Labeled Cells.

imaging the labeled cells : QDs can be imaged using any type of fluorescence microscope, including epifluorescence, confocal and multiphoton. However, unlike with conventional fluorophores, a single wavelength of light can be used to excite several different color QDs. Because most commercially available QDs emit in the green to red region of the visible spectrum, a microscope capable of providing an excitation beam (from lamp or laser) in the UV to blue region of the spectrum and capable of resolving multiple emission wavelengths could be used. As QDs are better excited by UV light, fixed cells can be imaged using a UV light source. To minimize UV-induced photodamage, live cells should be imaged using a blue (wavelength >400 nm) excitation light. For 2-photon imaging, excitation at 800 nm is optimal, but any wavelength of light between 700 and 1,000 nm could be used^ref. The choice of emission filter will depend on the emission spectrum of the QD in use. During the course of imaging for all live cell studies, it is recommended that the cells be maintained at 37 °C and not at room temperature (15–25 °C). Although QDs are highly photostable, long exposures to an excitation light source or exposure to UV light can lead to photodamage to the labeled cells. Thus, during long-term imaging, attempts should be made to minimize the length of exposure to excitation light and avoid the use of UV excitation. For multicolor imaging, instead of taking sequential images for each color QD, the emission from each color QD should be acquired simultaneously (if possible) using devices, such as dual view, quad view (Optical Insights), META detector (Zeiss) or AOBS (Leica), that allow simultaneous resolution of different-color QDs.

^ref

generalized labeling of live cells : unmodified hydrophilic QDs can be used for applications that involve nonspecifically tagging cells for long-term or multicolor imaging. To tag cells at their surface, QD avidin bioconjugates can be used^ref. Because QDs are membrane impermeant, depending on the cells under study, one of the following alternative approaches can be used to tag cells with QDs. In all cases, the labeled cells may be observed as described in Imaging the Labeled Cells.

endocytic labeling leads to localization of the QDs to endosomes4. Incubate cells capable of endocytosis with 1 mM DHLA-capped QDs for 2–3 h in the appropriate growth medium. Remove the excess QDs by washing the cells several times with growth medium or an appropriate buffer. Because different cells have different rates of endocytosis, the optimal time for endocytic loading should be determined for each cell type and cell line being used. Although DHLA-capped QDs are not toxic to cells when delivered by endocytosis^ref or by lipid-based reagent^ref, QDs with different chemical coats may cause toxicity. Therefore, when used for the first time, QDs must be tested for any deleterious effects.
labeling with cationic lipid-based reagents also allows efficient and rapid delivery of QDs into the cytosol^ref. Incubate 100 pmol of DHLA-capped QDs in 100 ml of serum-free medium containing a lipid-based transfection reagent appropriate for transfecting 3 mg of plasmid DNA. Add this mixture to cells growing in serum-free or complete medium and incubate for 1–2 h. QDs that do not have a negatively charged surface coating will give poor loading efficiency. Trying different transfection reagents and different ratios of mixing for the transfection reagent and QDs may improve the loading efficiency and reduce cell death.
labeling using an amphipathic carrier peptide can facilitate the uptake of cell-impermeant macromolecules. The peptide Pep1 (KETWWETWWTEWSQPKKKRKV) has been used to transport proteins^ref and QDs into cells^ref. Incubate cells for 1 h in serum-free medium containing a preformed Pep1-QD complex (10 mM peptide and 100–500 nM QDs in 100 ml of serum-free medium). Wash the cells two or three times with PBS or serum-free medium to remove extracellular QD-peptide complex. Although the use of this approach allows cellular labeling using fewer QDs, it is still dependent on the endocytic ability of cells, as labeling is abrogated in cells incubated at 4 °C (J.K. Jaiswal and S.M. Simon, unpublished observations). This mode of QD delivery by Pep1 is consistent with TAT and polyarginine peptide–mediated protein delivery, which also occurs by endocytosis^ref.
labeling of the cell surface allows labeling of both prokaryotic and eukaryotic cells. Wash the cells free of growth medium using PBS, then incubate them in a solution of 1 mg/ml Sulfo-NHS-SS biotin in PBS either for 30 min at 4 °C or for 5 min at 20–25 °C. Quench the excess biotin by washing the preparation with TBS (pH 7.4), then incubate the cells for 10 min in a serum-free medium containing 0.5–1 mM avidin-conjugated QDs. Remove the unbound QDs by repeated washing with PBS. Biotinylation of cell membrane and subsequent binding of avidin-conjugated QDs could affect the cell-surface properties, such as adhesion, cell-cell interaction and signaling. This concern must be taken into consideration when this approach is employed for functional studies using these cells.
microinjection is also useful for localized labeling of neurons or other cells in situ. This approach has been used to label isolated Xenopus eggs, which subsequently undergo normal development^ref. Resuspend QDs at a concentration of 1–10 pmol/ml in the appropriate buffer (for example, for mammalian cells, nuclear injection buffer can be used). Inject the desired amount of QDs into the cells. Allow the cells to recover before imagining.

label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest^ref

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. MRI technology can use perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' ¹H image places the cells in their anatomical context. Phenotypically defined dendritic cells (DCs) can be labeled with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. Labeled DCs were observed into tissue or intravenously in mice and then tracked the cells in vivo using ¹⁹F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo^ref.

Technical difficulties in tracking endogenous CD4 T lymphocytes have limited the characterization of tumor-specific CD4 T cell responses. Using fluorescent MHC class II/peptide multimers, we defined the fate of endogenous Leishmania receptor for activated C kinase (LACK)-specific CD4 T cells in mice bearing LACK-expressing TS/A tumors. LACK-specific CD44^hi62L^lo CD4 T cells accumulated in the draining lymph nodes and had characteristics of effector cells, secreting IL-2 and IFN-gamma upon Ag restimulation. Increased frequencies of CD44^hi62L^lo LACK-experienced cells were also detected in the spleen, lung, liver, and tumor itself, but not in nondraining lymph nodes, where the cells maintained a naive phenotype. The absence of systemic redistribution of LACK-specific memory T cells correlated with the presence of tumor. Indeed, LACK-specific CD4 T cells with central memory features (IL-2⁺IFN-g^-CD44^hi62L^lo cells) accumulated in all peripheral lymph nodes of mice immunized with LACK-pulsed dendritic cells and after tumor resection. Together, our data demonstrate that although tumor-specific CD4 effector T cells producing IFN-g^- are continuously generated in the presence of tumor, central memory CD4 T cells accumulate only after tumor resection. Thus, the continuous stimulation of tumor-specific CD4 T cells in tumor-bearing mice appears to hinder the systemic accumulation of central memory CD4 T lymphocytes^ref.