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Materials

Cells of interest

1% (v/v) methanol-free formaldehyde (Polysciences) in PBS (appendix2a), pH 7.4 (primary fixative)

PBS (APPENDIX 2A)

70% ethanol (secondary fixative)

5x TdT reaction buffer (see recipe)

TdT in storage buffer (both from Roche Diagnostics), 25 U in 1 |l

10 mM cobalt chloride (CoCl2; Roche Diagnostics)

Rinsing buffer: PBS with 0.1% (v/v) Triton X-100 and 0.5% (w/v) BSA

FITC-conjugated anti-BrdU MAb in PBS (appendix 2a; see recipe)

PI staining buffer: PBS with 5 |g/ml PI and 200 |g/ml DNase-free RNase

Flow cytometer with 488-nm excitation and filters for collection of green and red fluorescence

Fix cells

1. Fix 1-5 x 106 cells in suspension 15 min in 1% methanol-free formaldehyde in PBS, pH 7.4, on ice.

2. Centrifuge 5 min at 200 x g, 4°C, resuspend cell pellet (~2 x 106 cells) in 5 ml PBS, centrifuge 5 min at 200 x g, 4°C, and resuspend cells in 0.5 ml PBS.

3. Add the 0.5-ml cell suspension from step 2 to 5 ml ice-cold 70% ethanol.

The cells can be stored several weeks in ethanol at -20°C.

4. Centrifuge 5 min at 200 x g, 4°C, remove ethanol, and resuspend cells in 5 ml PBS. Repeat centrifugation.

Stain cells

5. Resuspend the pellet (not more than 106 cells) in 50 |l of a solution that contains:

10 |l 5x TdT reaction buffer

2.0 |l 2 mM BrdUTP stock solution

5 |l 10 mM CoCl2 solution

6. Incubate cells in this solution 40 min at 37°C.

Alternatively, incubation can be carried out overnight at 22° to 24°C.

7. Add 1.5 ml rinsing buffer and centrifuge 5 min at 200 x g, room temperature.

8. Resuspend cells in 100 | l FITC-conjugated anti-BrdU MAb solution.

9. Incubate 1 hr at room temperature or overnight at 4°C. Add 2 ml rinsing buffer and centrifuge 5 min at 200 x g, room temperature.

10. Resuspend cell pellet in 1 ml PI staining solution containing RNase. Incubate 30 min at room temperature in the dark.

11. Measure cell fluorescence on the flow cytometer, using 488-nm excitation (or a mercury arc lamp with a BG12 filter). Collect green FITC-anti BrdU MAb fluorescence at 530 ± 20 nm and red PI fluorescence above 600 nm.

Nucleic Acid Analysis

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