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 Intracellular staining of cytokines (BD Pharmingen)
A) Materials:
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BD Perm/Wash Buffer 10x (dilute in dd H2O before use)
BD Cytofix/Cytoperm
PMA [100 g/mL]
Ionomycin [200 g/mL]
Brefeldin A [10 mg/mL] or Monesin [2 mM]
B) Procedure:
1. Count cells and ressuspend in media at 1x106 cells/ mL/ tube.
2. Add PMA at final concentration of 100 ng/mL (stock: 100 g/mL) and Ionomycin at
final concentration of 200 ng/mL (stock: 200 g/mL) for 2 h at 37 oC.
3. Add Brefeldin A at 10 g/mL (or Monensin at 2 M) and incubate for further 2 h at 37
o
C.
4. Centrifuge and wash cells with cold PBS (3x).
5. Ressuspend cells with the leftover of PBS (Vortex).
6. Add FcBlock for 20 min at RT.*
7. Stain for surface markers for 10 min at R. T.
8. Centrifuge and wash cells with cold PBS (3x).
9. Ressuspend cells with the leftover of PBS (Vortex).
10. Add 100 L/tube of BD Cytofix/Cytoperm solution and incubate for 20 min at R.T in
the dark.
11. Centrifuge for 5 min at 0,5 rcf* and wash cells with BD Perm/Wash buffer (2x) or
100 L of Permeabilization buffer.*
12. Ressuspend cells with the leftover of BD Perm/Wash buffer.
13. Add 10 L of anti-cytokine antibody (1:100) diluted in BD Perm/Wash buffer at R.T.
for 20 min in the dark.
13. Centrifuge and wash cells with BD Perm/Was buffer (2x).
14. Ressuspend cells in 100 L of FACS staining buffer (PBE) and do flow cytometric
analysis.
*NOTE: steps “underlined” are our lab suggestions.