Generation of DCs

  • Complete RPMI: RPMI 1640 w/ L-Glutamine and 25 mM HEPES; +10% FCS; +nonessential amino acids (from 100x stock); + Sodium Pyruvate (from 100x stock) +penicillin/streptomycin (from 100x stock); + 55 uM B-ME (from 1000x stock).
  • Recombinant human Flt3L-Fc (BioXcell; 0.5 mg/ml stock; use at 1/5000 dilution)

 

  1.  CO2 euthanasia, remove femurs/tibias and clean off muscle. Cut off the top end of each bone.
  2. Puncture hole in 0.5 ml thin wall tube with 20 to 22 gauge needle (gauge size is important). Place bone facing down in the 0.5 ml tube, and then place tube into a 1.5 ml tube containing 100 ul of RPMI. Spin down for 1 min at 4000 G in a microfuge.
  3. Recover cells, centrifuge, and resuspend in 10 mls of BD PharmLyse (1/10 from 10X stock using ddH2O). Spin down immediately for 5 min.
  4. Remove supernatant and resuspend in 20 mls of DC media
  5. Count and dilute to 2x106 cells/ml in media containing 100 ng/ml Flt3L-Fc. Expect 3-4 x107 cells per mouse.
  6. Plate cells at 5 ml/well in 6 well plates.
  7. Culture for 8 days without disturbing. Expect 0.8-1.06 per ml with >90% purity.

 

Stimulation of DCs:

  1. Remove cells in suspension by pipetting. Rinse once with warm PBS to remove loosely adherent cells.
  2. Count cells and resuspend at 1x106 per ml with 100 ng/ml Flt3L-Fc
  3. Transfer 500ul of cells to each well of a 48-well tissue culture plate.
  4. Add stimulating/suppressing agents as desired:
    • Murine IL-10 (Peprotech): 10 ug/ml stock: Use at 20ng/ml (1/500 = 10U/ml)
    • Murine IL-4 (Peprotech ) 10 ug/ml stock: Use at 20ng/ml (1/500 = 10U/ml)
    • Murine IL13 (Peprotech) 10ug/ml stock: Use at 40ng/ml (1/250 = 10U/ml)
    • Human TSLP (Peprotech; 10ug) 10ug/ml stock: Use at 20 ng/ml (1/500)
    • Murine GM-CSF (Peprotech) 10ug/ml stock: Use at 10ng/ml (1/1000=200U/ml)
    • IFNG (Peptrotech): 10 ug/ml stock. Use at 40 ng/ml (1/250)
    • Anti-CD40 (FGK45): Use at 10 ug/ml.
    • LPS-EB Ultrapure (InvivoGen; tlrl-3pelps): TLR4 activator, 10ng – 10ug/ml range, use at 100 ng/ml (1/10,000 dilution of 1 mg/ml stock
    • Pam3CSK4 (InvivoGen; tlrl-pms): TLR1/2 activator, 1-300 ng/ml range, use at 100 ng/ml (1/5,000 dilution from 500 ug/ml stock)
    • Poly(I:C)-LMW (InvivoGen; tlrl-picw): TLR3 activator, 30ng – 10ug/ml range, use at 1 ug/ml (1/5,000 dilution of 5 mg/ml stock)
    • CpG ODN2395 (InvivoGen; tlrl-2395): TLR9 activator, 1-10 ug/ml range, use at 1ug/ml (1/1000 of 1 mg/ml stock)
    • Imiquimod (InvivoGen tlrl-imq): TLR7 activator, 1-5 ug/ml range, use at 1 ug/ml (1/1000 of 1 mg/ml stock)
  5. Incubate for 24 hrs.
  6. Add Brefeldin A for final 4 hrs (at hour 20) if doing intracellular staining.
  7. Harvest cells and supernatant for analysis. Wash 1x with Versene to remove adherent cells (mature DCs).