DC (Dendritic-Cell) Isolation - Method-Protocol (Mar/13/2002 )

Does any one have a good idea of how many DCs should be recovered from a normal mouse spleen?

for isolation of DC from mouse spleen.the best protocol is described in RM steinman's pioneering paper of 70's in Journal of experimental medicine. u can modify it according to ur need........

preparation of DC from mouse bone marrow (BMDC)

kill mouse (I've always used BALB/C, but the protocol is accurate also for other stems), e.g. by cervical dislocation
cleanly excise both femurs and remove excess tissue
cut off both ends of each femur and flush out bone marrow with a 27G needle (attached to a syringe with DC complete medium = DCCM)
resuspend gently to dissolve cell aggregates
pellet suspension for 5 min at 1300 - 1500 rpm and resuspend then in 1 ml DCCM
perform erythrocyte lysis with dH2O (3 ml for exactly 15 sec) and "stop" lysis by adding 3 ml 0.3M NaCl (i/c)
centrifuge again and resuspend in suitable vol DCCM
seed into 10 cm diameter bacteriological grade dishes (in 10 ml) - I recommend Falcon-1001 or Greiner)
keep at 37°C, 7% CO2 and feed every 3 days by adding 10 ml fresh DCCM or - dep on the cell density - split on day 6 into 2 dishes (10 ml each + 10 ml fresh medium)
at day 10, app. 10e7 immature DC can be harvested by pipetting (supsension cells)

note: cells will mature automatically by prolonged culture periods (use between day 10 and max. 14 for experiments)

DCCM:
RPMI-1640
5% FCS
2 mM L-glutamine
1% pen/strep (sigma)
200 µM ß-mercaptoethanol
10% GM-CSF conditioned supernatant (produced by GM-CSF-secreting cell line) OR 1-2 ng/ml rGM-CSF