100um cell strainer filter

Fresh iliac crest bone marrow aspirates from healthy human donors were collected at the Perelman School of Medicine, University of Pennsylvania, in heparinized syringes and transported to the lab for further processing within 4 h of sample collection. Cells were isolated from bone marrow aspirate samples by placing sample on top of a 100um cell strainer filter (Corning) followed by mechanical dissociation using the flat end of a 5 ml syringe (BD Biosciences). Cells were washed with DPBS and then underlaid with room temperature 96% Ficoll (GE Healthcare) prior to centrifugation at 400 × g for 30 min with 3 acceleration and no brake. Following centrifugation, mononuclear cells were harvested and washed with DPBS before RBC lysis was performed in ACK Lysis Buffer (Gibco). Cells were resuspended in cRPMI prior to cell counting. All samples were processed and stored at room temperature unless otherwise noted.

Whole femur bone marrow was collected from rhesus macaques <2 h post-necropsy. Whole femur marrow was harvested into sodium citrate containing tubes (BD Biosciences). Cells were isolated from bone marrow samples by placing sample on top of a 100 um cell strainer filter (Corning) followed by mechanical dissociation using the flat end of a 5 ml syringe (BD Biosciences). Cells were washed with DPBS and then underlaid with room temperature 96% Ficoll (GE Healthcare) prior to centrifugation at 400 × g for 30 min with 3 acceleration and no brake. Following centrifugation, mononuclear cells were harvested and washed with DPBS before RBC lysis was performed in ACK Lysis Buffer (Gibco). Cells were resuspended in cRPMI prior to cell counting. All samples were processed and stored at room temperature unless otherwise noted.