The principle method for generating BM-DC with GM-CSF was adapted from previous publications (Inaba et al., 1992a and Inaba et al., 1992b; Scheicher et al., 1992; Inaba et al., 1998).
Modifications were as follows. Instead of 24-well plates, bacteriological petri dishes with 100 mm diameter were used (Falcon, No. 1029/Becton Dickinson, Heidelberg, Germany). Cell culture medium (R10) was RPMI-1640 (GIBCO BRL, Eggenstein, Germany) supplemented with Penicillin (100 U/ml, Sigma, Deisenhofen, Germany), Streptomycin (100 μg/ml, Sigma), -glutamin (2 mM, Sigma), 2-mercaptoethanol (50 μM, Sigma), and 10% heat-inactivated and
filtered (0.22 μm, Millipore, Eschborn, Germany) FCS (PAA, Cölbe, Germany).
6At day 0 BM leukocytes were seeded at 2×10 per 100 mm dish in 10 ml R10 medium containing
6200 U/ml (=20 ng/ml) rmGM-CSF (5×10 U/mg; Peprotech/Tebu, Frankfurt, Germany).
At day 3 another 10 ml R10 medium containing 200 U/ml rmGM-CSF were added to the plates. At days 6 and 8 half of the culture supernatant was collected, centrifuged, and the cell pellet resuspended in 10 ml fresh R10 containing 200 U/ml rmGM-CSF, and given back into the original plate.
At day 10 cells can be used already or culture can be continued to further reduce granulocyte contamination. Then plates were fed as at day 6 and 8, but the dose of rmGM-CSF in the fresh 10 ml R10 medium contained only 30–100 U/ml. For complete maturation the day 10 non-adherent
cells were collected by gentle pipetting, centrifuged at 300×g for 5 min at RT, and resuspended in
10 ml fresh R10 into a fresh 100 mm tissue culture plastic dish (Falcon, No. 3003) containing 100 U/ml rmGM-CSF and tumor necrosis factor α (TNF-α, Peprotech) at 500 U/ml or
lipopolysacchride (LPS, Sigma) at 1 μg/ml. Cells were then cultured for 1 or 2 more days. Culture supernatant from a cell line transfected with the murine GM-CSF gene was used equally well instead of rmGM-CSF (Zal et al., 1994). 10% GM-CSF supernatant were found to generate
BM-DC equally well to 200 U/ml rmGM-CSF.