Intracellular Cytokine or Protein Analysis

Determine whether a cell in a mixed population expresses a certain cytokine or protein internally, by fixing and permeabilizing cells allowing conjugated antibodies to pass through the cell membrane.

  1. Stain surface antigens as described in the Cell Surface Staining protocol.
  2. After incubation wash and discard the supernatant and vortex to dislocate the pellet.
  3. Fix cells by adding 100µl of Fixation buf fer and vortex.
  4. Incubate in the dark for 30min. Without washing add 2mL of Permeabilization buffer to each tube.
  5. Centrifuge sample at 1200 rpm at room temperature for five minutes and discard supernatant.
  6. Resuspend pellet in 100µl of Perm buffer, and add the recommended amount of fluorochrome-labeled antibody for detection of intracellular antigens to the cells.
  7. Incubate for 30min in the dark at room temperature.
  8. Add 2mL of Perm buffer to each tube. Centrifuge samples at 1200 rpm at room temperature for 5 minutes, then discard the supernatant.
  9. Wash by adding 2mL FACS buffer to the sample and centrifuging again.
  10. Resuspend stained cells in 300-500µl of FACS buffer.
  11. Send samples to AVIVA for analysis by flow cytometry.