BMDM isolation and culture

Materials:

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)

BMDM growth medium:

IMDM+10%FBS+15%L929 SN (or 10ng/ml GM-CSF)

Protocol:

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 500 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 500 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.

Remove BMDM from dish (it is better to use petri dish for BMDM culture)

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