PROCEDURE C
Grow up cell cultures and expose to appropriate
experimental conditions.
Trypsinize (0.25%, GIBCO) cells and collect
in 15-ml conical tubes. At this point, many procedures call for the addition
of buffer with serum to inactivate the trypsn;
RPA-SMC apparently do not require this step.
Centrifuge 5 min at 1,500 rpm
Decant supernate and resuspend in 1 ml of
PBS (pH 8)
Remove 30µl from each sample and place
in microfuge tubes with 30µl of 0.4% trypan blue. Mix gently.
Count viable cells using a hemacytometer and
calculate # cells/ml/
Transfer volume of cell suspension necessary
for 1-5 x 106 cells to a centrifuge tube (microfuge, if possible)
Centrifuge 5 min at 1,500 rpm
Decant supernate and resuspend in 150µl
of 1x PBS (pH8)
Add 375 µl of FACS buffer to each tube.
Mix
Add 250µl of RNAse to each tube.
Add 100µl of 0.2% Triton-X 100 to each
tube. Mix
Incubate at 37C in 5% CO2 in air for 30 min
Add 100 µl of propidium iodide solution
to each tube. Cover with aluminum foil.
Incubate at R.T. for 30 min.
Transfer cell suspensions to Falcon 2063 tubes
for FACS analysis. Samples may be stored O.N. at 4C
| FACS buffer: (250 ml: store at R.T.) | |
| 100 mM sodium acetate | 3.4 g NaC2H3O2 |
| 5.4 mM EDTA | +0.5 g EDTA |
| pH to 5.2 | |
| qs 250 ml | |
| RNAse: (for 1 ml; prepare fresh) | |
| 700 U/ml RNAse | 10 mg of 70 U/mg RNAse |
| +1 ml FACS buffer | |
| 0.2% Triton-X 100: (for 50 ml) | 1 ml undiluted Trition-X 100 |
| +49 ml FACS buffer | |
| propodium iodide solution: (for 2ml) | 1 mg propodium iodide |
| + 2 ml FACS buffer | |
[ Adapted from R.L. Vendor, et. al., Reduced oxygen tension induces
pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s),
Amer. Rev. Resp. Dis., 135:622-7, 1987.]