BMDM isolation and culture
1. IMDM +10%FBS
2. PBS +2% FBS
3. L929 cell supernatant or GM-CSF
4. 70um cell strainer
5. 21g or 23g needles
6. surgery tools
7. NH4Cl solution (Stemcell Laboratory)
IMDM+10%FBS+15%L929 SN (or 10ng/ml GM-CSF)
1. Isolate femur and tabular bone from B6 mice, rinse off hairs before cutting open the bone
2. Use 23g or 21g needle on 10ml syringe filled with cold PBS+2% FBS (3-5ml for one mouse) to flush marrow from the bone
3. Pass marrow through 23g or 21g needles 4-6 times to separate cells
4. Pass cell through a 70um cell strainer to remove cell clumps, bone, hair or other tissue
5. Add 3 vol (~15 ml) of NH4Cl solution, incubate on ice for 10 min to remove red blood cells
6. Spin down, 2000rpm (or 900 x g) for 5 min
7. Resuspend cell pellet with cold PBS+2% FBS (20-50ml, depending on the cell quantity). Count the cells.
8. Spin down, 2000rpm (or 900 x g) for 5min.
9. Resuspend cells at 1-1.5x10^6/ml with BMDM growth medium: IMDM + 10% FBS + 15% filtered L-929 medium.
10. Seed cells at 1-1.5x10^6/ml in 12 well plates. 15ml per 10cm plate. (Or, Keaton: one mouse for 50ml culture).
11. Change fresh growth medium on day 3.
12. BMDM can be used for further study on day 7.
DPBS(free Mg,Ca)/EDTA (0.5M) COLD
1. Wash BMDM with PBS (cold) after removal of medium
2. Add DPBS(Ca-, Mg-) + 5mM EDTA (cold)
3. Place cells on ice for 20-30 minutes (check every 10-15 min), tap the plate
Pipetting cells to collect! These are good for FACS analysis