The protocol below is designed for general purposes. You must adapt dilutions and times depending on your specific requirements.
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1. Count 300.000 cells per experiment and, put them on ice and resuspended in FACS buffer
2. Use a 96-well plate (v-bottom) on ice and place 300.000 on each well.
3. Centrifuge the plate at 1400 rpm for 5 to 6 minutes in cold and carefully pour the supernatant. Resuspend in 200 μl / well of fresh FACS by using vortex or multichannel pipette.
4. Repeat the wash twice in identical conditions.
(Shoud you wish to detect an intracellular protein, follow from here, if not, you can go to step 8)
5. Resuspend cells in 200 μl of fixation buffer and incubate for 20 minutes at room temperature.
6. Centrifuge at 1500 rpm for 7 minutes.
7. Incubate for 10 minutes with 200 μl of permeabilisation buffer.
Flow cytometric patterns of cardiomyocytes stained with Rho123, DioC6(3) and CMXRos. Mathur A et al. Cardiovasc Res 2000;46:126-138
8. Add the antibodies of interest at the appropriate dilutions in 50 μl FACS buffer or 50 μl of FACS.
9. Homogenize and incubate for 1 hour at 4 ºC.
10. Carry up to 200 μl with FACS buffer and wash 3 times at 1400 rpm for 5 minutes in cold.
11. Add the secondary antibodies at the appropriate dilutions in 50 μl FACS buffer or 50 μl and incubarte for 30 minutes in the dark at 4 ºC.
12. Centrifuge at 1500 rpm for 7 minutes , remove supernatant and resuspend in 200 μl of FACS buffer.
At the time of analysis, you should resuspend the cells in 400 μl of FACS buffer using the appropriate tubes for the instrument.
Fixation buffer: 4% paraformaldehide in FACS pH 7.5. (Store at 4ºC)
Permeabilisation buffer: 0.1% Triton X-100 in FACS.