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Flow Cytometry Protocol
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This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.
Preparation- Harvest and wash the cells and determine the total cell number.
- Resuspend the cells to approximately 1x106 cells/mL in ice cold PBS. Add the appropriate volume of paraformaldehyde to obtain a final concentration of 4%.
- Fix for 10 minutes in a 37°C water bath.
- Wash cells twice with PBS containing 0.5% BSA.
Note: For extracellular staining with antibodies that do not require permeabilization, proceed to step 8; for intracellular staining, proceed to step 5.
- Add ice-cold 90% methanol (approximately 1mL per 1x106 cells) and vortex.
- Permeabilize for a minimum of 10 minutes on ice.
- Wash cells twice with PBS containing 0.5%BSA.
- Resuspend 1x106 cells in 100μL PBS containing 0.5%BSA.
- Add the primary antibody and incubate for 1 hour at room temperature (RT).
- Wash cells twice with PBS containing 0.5%BSA.
Note: If using a fluorescent primary conjugated antibody, skip to step 14. - Resuspend cells in fluorochrome conjugated secondary antibody diluted in PBS containing 0.5% BSA.
- Incubate for 30 minutes at RT.
- Wash cells twice with PBS containing 0.5% BSA.
- Resuspend cells in 0.5mL PBS and analyze on flow cytometer.