Injekt

C57BL/6 mice that expressed luciferase from Mx2 promoter (Mx2 luc) were euthanized by CO 2 asphyxiation 31 . Femur and tibia bones from the hind legs were isolated and washed in a petri plate filled with ice cold RPMI (Rosewell Park Memorial Institute) medium (Sigma, Germany) supplemented with 1% Penicillin/Streptomycin (Gibco, Germany) and 1% Glutamine (Gibco, Germany). The bones were submerged in 70% ethanol for 1 minute and then transferred to ice cold RPMI media. Then the joints were cut off and the bone marrow was flushed out with ice cold RPMI medium using a syringe (Injekt, B. Braun Melsungen AG, Germany) with a 26G hypodermic needle (Sterican, B. Braun Melsungen AG). Cell suspensions were collected in 50 ml Falcon tubes and centrifuged at 1500 rpm for 5 minutes.
Supernatants were discarded and cell pellets were resuspended in erythrocyte lysis buffer. ACK lysis buffer (1 ml per mouse) was added, followed by incubation at room temperature for 1.5 minutes. Then 10 ml of RPMI medium was added and intact cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The supernatant was discarded and cells were suspended in 10 ml of RPMI media. Cells were filtered through 100 μm strainer (BD falcon, Germany) and counted using an automated cell counter (Beckman Coulter, Germany).