The following protocol, courtesy of PharMingen, is for intracellular cytokine staining using the formaldehyde/saponin method.

Reagents

  • Fluorochrome-conjugated anti-cytokine monoclonal antibody
  • Fluorochrome-conjugated antibody to cell surface antigen
  • Isotype control antibody
  • Staining Buffer:
    • Dubecco's PBS (DPBS)
    • 1% heat-inactivated FCS
    • 0.1% (w/v) sodium azide
    • Adjust buffer pH to 7.4 - 7.6, filter (0.2µm pore membrane), and store at 4°C.
  • Fixation Buffer:
    • Dulbecco's PBS (DPBS)
    • 4% (w/v) paraformaldehyde
    • Add paraformaldehyde to DPBS and warm in 56°C water bath (in fume hood) until paraformaldehyde dissolves (about 60 minutes). Adjust buffer pH to 7.4 - 7.6 and store at 4°C.
  • Permeabilization Buffer:
    • Dulbecco's PBS (DPBS)
    • 1% heat-inactivated FCS
    • 0.1% (w/v) sodium azide
    • 0.1% (w/v) saponin
    • Adjust buffer pH to 7.4 - 7.6 and filter (0.2µm pore membrane). 

Staining

  1. Stain 106 cells in 50 ul of staining buffer with a fluorochrome-conjugated antibody specific for a cell surface antigen (30 min., 4°C).
  2. Wash cells with staining buffer and pellet by centrifugation (250 x g).
  3. Thoroughly resuspend and fix cells with 100µl of fixation buffer (20 min., 4°C).
  4. Wash cells in staining buffer and pellet.
  5. Thoroughly resuspend fixed cells in 50µl of permeabilization buffer.
  6. Add an optimal dose of fluorochrome-conjugated anti-cytokine antibody and incubate (30 min., 4°C).
  7. Wash cells in permeabilization buffer and pellet.
  8. Thoroughly resuspend cells in staining buffer and analyze the stained samples by flow cytometry.