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Viable Fluorescence Stains
The use of fluorescent dyes has evolved into a powerful tool for assessing cell function, viability, and proliferation. Because of the greater sensitivity, current fluorescent methods can often replace colorimetric assays for toxicity studies and high-throughput drug
Centering the condenser
Centering the annular diaphram
Adjusting the phase. (A) Diagram of how to adjust the phase rings. (B) View of the diaphragm. (C) Centering the annular rings. See text for for instructions on adjusting the phase.
Figure 6.8. (Continued).
screening. Moreover, viable fluorescent tracers can be used to follow events in long-term transplantation of cells, to study dye translocation across cell membranes, to follow the movements of labeled cells in culture, and to investigate events during the cell cycle (Eidelman and Cabantchik, 1989; Johnson et al., 1980). Fluorescent latex microspheres can also be used as microinjectable cell tracers as well as markers for cellular antigens and for the study of phagocytotic processes.
Dil and DiO (Molecular probes, D-282, D-275) are lipophilic dyes that have been used extensively for tracing the movement and integration of neuronal cells both in vitro and in vivo (Fig. 6.10). Cells seem to tolerate high concentrations of these dyes and remain viable for extended passages over time in culture (Kuffler, 1990; Serbedzija et al., 1990).
Fluorescent cell linker compounds are also available from a number of vendors (i.e., PKH26, a red fluorescent cell linker from Sigma). These compounds have been found to be useful for cell labeling and tracking both in vitro and in vivo by incorporating aliphatic reporter molecules into the cell membrane by selective partitioning (Horan and Slezak, 1989). An esterase substrate, such as calcein, can also be used as a live cell stain, as it is retained within the cell for up to 3 hr at 37jaC.
Fluorescent double stain of human Schwann cells in secondary cultures. The cell nuclei were stained with propidium iodide, which fluoresces red. The cell cytoplasm was stained for GAP (left) or S100 (right) using a green fluorescent-tagged antibody. Yellow nuclei are cells that stain both red and green. Any cells that do not stain with the specific markers will retain red nuclei.
Differentiation and integration of viable dye-marked NEP cells (upper left) into neonatal rat brain. Cultured cells were labeled with the vital fluorescent dye Dil and injected near the cortical surface in newborn rat brains in the area shown in the lower left brain section. After 2 days, the animals were killed and the brain sectioned and observed with fluorescent microscopy. The NEP cells have migrated into the brain and differentiated into cells resembling cortical neurons (seen at low power on the right and higher power on the upper left).
Labeling Cells with a Fluorescent Viable Cell Dye Materials
1. Cell cultures, 2 x 107 cells/sample
2. Growth medium, serum free
3. Growth medium (serum free) with 0.1% BSA
4. 15-ml conical polypropylene centrifuge tubes
5. Clinical centrifuge
6. Microscope with epifluorescence
7. Pkh 26 kit (sigma)
1. Trypsinize and neutralize cell suspension.
2. Count the cells by hemacytometer or electronic counter.
3. Resuspend 2 x 107 cells (2x stock) in serum-free medium and centrifuge at low speed (400 xg) for 5 min to obtain a loose pellet.
4. Prepare 4 x 10 "C 6M dye solution with the diluent supplied with the kit (this is a 2x stock concentration). Add 1 ml to a 15-ml conical polypropylene tube and set aside.
5. Aspirate the supernatant from the pellet, allowing no more than 25 ^l to remain on the pellet.
6. Tap the pellet in the remaining supernatant to loosen the cells.
7. Add 1 ml of the diluent to the cells, gently resuspend, and add this 1 ml to the tube that has the l-ml dye solution.
8. Gently resuspend the sample.
9. Incubate at room temperature for 7 min.
10. Add 2 ml of F12.DME plus (0.1%) BSA. Allow to stand 1 min.
11. Add 4 ml of growth medium to the tube.
12. Centrifuge for 10 min at 400 xg.
13. Aspirate supernatant, transfer the pellet to a new 15-ml conical tube, and wash by centrifugation 3x in 10 ml of F12/DME plus 5% BSA.
14. Resuspend cells to the desired concentration for in vivo injection (107 cells/500 ^l per 100 ^l) or in vitro assessment (106 cells/60-mm plate), in growth medium.
Immunohistochemistry can provide a rapid method for demonstrating the presence and localization of an antigen. Double labeling allows the simultaneous detection of two antigens, thus offering more information about their relative distribution. If the antigen is highly localized, it may be possible to detect as few as 1000 molecules in a cell. With advances in antibody labeling, enhancing, and particularly cell-staining techniques, immunohistochemistry can be a powerful quantitative technique as well.