Flow Cytometry Protocol

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 1x10^6 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 immunostaining.

Permeabilization

  • Add ice-cold 90% methanol (approximately 1mL per 1x10^6 cells) and vortex.
  • Permeabilize for a minimum of 10 minutes on ice.

Immunostaining and Analysis

  • Wash cells twice with PBS containing 0.5%BSA.
  • Resuspend 1x10^6 cells in 100μL PBS containing 0.5%BSA.
  • Add the primary antibody at the recommended dilution of 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 the secondary antibody

  • Resuspend cells in fluorochrome-conjugated secondary antibody diluted in PBS containing 0.5% BSA.
  • Incubate for 30 minutes at RT in the dark.
  • Wash cells twice with PBS containing 0.5% BSA.
  • Resuspend cells in 0.5mL PBS and analyze on the flow cytometer.
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