1. Add IMDM equal to the total BMA volume to the EDTA tubes.
2. In 15/50ml conicals, add BMA/IMDM mixture into each with an equal amount of Ficoll-Paque (i.e. total BMA/IMDM volume is 14ml, place 3.5ml Ficoll & 7ml BMA/IMDM into two conicals). Note, tilt the 15ml conical to a 45O angle and layer the BMA/IMDM on top of the Ficoll (Don’t let the BMA mix with the Ficoll).
3. Centrifuge the 15/50ml conicals for 25 minutes with no brake at 1400rpm and 4C.
4. Carefully remove conicals from centrifuge and notice the layers. From bottom to top: RBCs, clear ficoll, cloudy interphase cells, and pink plasma + IMDM.
5. Using a glass pipette, collect the interphase cell layer avoiding Ficoll and place in a new 15ml conical.
6. Fill to the 14ml line on the conical with Rinsing Buffer and centrifuge 5min at 1500rpm and 20C.
7. After centrifuge, aspirate the supernatant and resuspend the pellet in 10ml of Rinsing Buffer.
8. Place a sterile pre-separation filter on top of a new 15ml conical.
9. Pre-wet the filter with 300ul of Rinsing Buffer, then filter the re-suspended cells.
10. Place 10ul of this re-suspension into a microcentrifuge tube.
11. Record total amount of resuspension before spinning.
12. Spin 15ml conical for 5min at 1500rpm and 20C.
13. In a 1ml cryovial, add 10ul AO/PI and 10ul aliquot of the BMMCs.
14. Place 10ul on hemocytometer and count live:dead, then record. Note, be sure to multiple by total volume of BMMCs
15. Remove supernatant, re-suspend in 1.5 mL of Freeze Media for approximately every 5E6 cells and aliquot into cryovials. Note, before use you must thaw Freeze Media in H2O bath.
16. Label each cryovial with barcoded label.
17. Store in cryovials of ~1e7 total cell aliquot(s).
18. Allow to Freeze slowly in a freezing chamber at -80C for 24 hours, then place the vial in Liq Nitrogen for long term storage.
19. Complete the processing and sample documentation sheet(s).