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9. Radioactive waste bags
10. 7-ml polypropylene scintillation vials Procedure
1. Trypsinize the cultures as previously described and take a cell count.
2. Plate 5 x 103 to 1 x 104 cells/well in growth medium, 2 ml/well.
3. Incubate the cultures for 24'C72 hr (this will depend on the cell type).
4. Place a sheet of absorbent plastic-backed paper (e.g., Benchcote) in the tissue culture hood.
5. Prepare assay medium: Growth medium containing 1 ^Ci/ml [3H]thymidine. Dispose of all pipettes and pipette tips in a radioactive waste bag.
6. Remove the plate from the incubator. Remove growth medium from the plate.
7. Add 2 ml of assay medium.
8. Incubate 3"C4 hr (this is for an established cell line that has a nominal doubling time of 24"C36 hr. Primary cell cultures or cell lines with >36 hr doubling times may require a longe^up to 24 hrjaincubation).
9. Remove the radioactive medium. Wash the plate 2x with serum free medium.
10. Add 1 ml/well cold 5% TCA, to extract residual thymidine. Note: TCA is a strong acid. Care should be taken when handling the crystals or solutions to avoid getting it on clothes or skin.
11. Repeat step 9. Tip the plate to remove as much TCA as possible.
12. Add 1 ml/well 0.1N NaOH.
13. Leave at room temperature until the cells are unattached and broken up (around 20"C30 min).
14. Resuspend the cells in the well with a 1 ml Pipetman. Note: The acid'Cbase ratios in the following steps are critical, as well as the order of addition.
15. Add 0.5ml of the cell suspension to a 7-ml scintillation vial.
16. Add 3.5 ml of scintillation fluid.
17. Add 200 ^l of 40% acetic acid.
18. Cap the vials and shake.
19. Count in an appropriate beta counter.
20. Dispose of the vials in a bag for radioactive waste, separate from all other radioactive waste, solid or liquid. (Individual institutions usually set up their own guidelines regarding the disposal of biohazard waste and radioactive compounds in compliance with city, state, and federal regulations.)
21. Be sure and treat any spills with solutions such as Count-Off.
High-Throughput Assays for Secondary Endpoints for Cell Number
It is difficult to develop a high-throughput assay for actually counting cell number. Therefore, many investigators use a secondary endpoint, which usually correlates well with increased or decreased cell number, as a screen. The thymidine incorporation assay described above is one such secondary endpoint. Others are based on colorimetric readouts such as crystal violet, a protein stain, or fluorometric readouts of dyes, such as calcein-AM, whose level in cells is proportional to esterase activity in the cells. Many of these assays are extremely sensitive and therefore can be used with cells in 96-well plates. The results can be automatically read on a plate reader, analyzed by an attached computer, and printed out in a form suitable for publication.
Measuring Cell Viability
Total cell number both in a tissue in vivo and in a culture dish in vitro is a balance between the rates of cell growth, or mitosis, and cell death. The ability to differentiate between live and dead cells is therefore important. Trypan blue dye exclusion (described in the section above on using the hemocytometer) is a rapid and reliable method that relies on the breakdown of membrane integrity, allowing the uptake of a dye that is normally membrane impermeant.
The following method is more sensitive in detecting damaged cells than trypan blue and it is quite easy to see the cells and do differential counts (Parks et al., 1979). A fluo-
rescent microscope with an appropriate filter, however, is required. Both acridine orange and ethidium bromide are DNA intercalating dyes and therefore mutagens. Handle and dispose with care.
Acridine Orange-Ethidium Bromide Viability Determination
1. Trypsinized and neutralized cell suspension
2. Ethidium bromide'Cacridine orange (EB-AC) stock solution (100x): ethidium bromide, 50 mg; acridine orange, 15 mg. Dissolve in 1 ml 95% ethanol and QS to a total volume of 45 ml in purified water. Store in 1-ml aliquots at "C20jaC.
1. Make a working solution of EB-AC by diluting a 1-ml aliquot of stock solution into 100 ml PBS. This can be stored, light tight, at 4jaC for up to 1 month.
2. Mix equal volumes of cell suspension: EB-AC.
3. With a Pasteur pipette or a Pipetman pipettor, add a drop (100 ^l) to a slide and cover with a coverslip.
4. Observe with an epifluorescent microscope using a filter excitation of 495 nm.
5. Count the live/dead cells with a tally counter. The live cells will fluoresce green and the dead cells will fluoresce orange.
6. Determine viability by calculating: (Live cells/Total cells counted) x 100.
More elaborate and accurate methods of differentiating live and dead cells include FACS, tunnel labeling, and electron microscopy. Methods for measuring apoptotic cell death are evolving very rapidly at this time (Mather and Moore, 1997; Moore et al., 1995, 1996), and more technological advances can be expected.