Cd41 clone mwreg30

Blood was collected from P14 mice by cardiac puncture at time of euthanasia in 50 mM EDTA containing Futan-75 to prevent complement activation. Collected samples were centrifuged for 10 min at 600 g. Supernatant was removed, and pellets resuspended in 1 mL citrate buffer containing 50 ng/mL PGE2 to prevent platelet activation. The resuspended samples were centrifuged for 10 min at 3,200 g. Supernatant was then discarded and erythrocytes lysed using ACK buffer (ACK Lysing Buffer, Gibco, ThermoFisher Scientific) for 4 min. The samples were then washed with PBS for 10 min at 3200 g. Supernatant was removed and samples containing circulating immune cells and platelets were resuspended in FACS buffer, incubated with anti-FcR antibody (clone 24G2) and stained with the following primary antibodies: Ly6G (clone 1A8), Ly6C (clone AL-21), CD11b (clone M1/70, eBioscience), CD62P (clone RB40.34) and CD41 (clone MWReg30). All antibodies were purchased from BD Pharmingen. After staining samples were washed twice in FACS buffer, fixed for 10 min at 4 °C in 1% paraformaldehyde, washed and resuspended in FACS buffer. The samples were acquired on a Fortessa X20 (BD Pharmingen) and analyzed with FlowJo software (TreeStar).