Cell culture and reagents

Culture of cryopreserved human PBMCs with IFN-β

1. Thaw a 1 ml vial of 10⁷ cryopreserved human PBMCs in a 37^oC water bath
   

2. Transfer the cells into a conical tube with 14 ml complete media

3. Centrifuge cells at 1400 rpm 4^oC for 7 minutes and discard supernatant

4. Resuspend cell pellet in 10 ml complete media

5. Count cells on a hematocytometer

6. Centrifuge cells at 1400 rpm 4^oC for 7 minutes and discard supernatant

7. Resuspend cells at a concentration of 2x10⁶ cells/ml 

8. Transfer 500 µl (10⁶) cells/well in a 24-well plate

9. Prepare a 2X solution of IFN-β in complete media (500U/mL)

10. Add 500 µl of 2X cytokine solution or 500 µl of complete media on top of cells (final concentration is 250 U/mL) 
    

11. Culture cells for 18-24h at 37^oC 5% CO₂ 
    

Complete media

- RPMI 1640                                                        

- 10% fetal bovine serum

- 1 mM sodium pyruvate

- 100 U/mL penicillin

- 0.1 mg/mL streptomycin

- 1% non-essential amino acids (Gibco)

- 0.1% mL 2-mercaptoethanol 

Cytokines

- Recombinant human IFN-β (Peprotech)

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