Hi,

Dilute the bone marrow with PBS at a ratio of 1:1 (add 50 ml of PBS to make it 100 ml) and use the ficoll to the original volume of bone marrow (50 ml). U can layer 20 ml of diluted bone marrow onto 10 ml of ficoll layer and centrifuge at 400g for 30 min at RT. aspirate the interphase (between the plasma and ficoll) which contains MNCs and wash once with PBS, centrifuge at 300 g at RT to pellet the cells.

You might want to try this (all steps at room temperature, i.e 20-25 oC)

1. Share 50 ml BM in 3 50 ml falcon tubes

2. Fill to the 40 ml mark each tube with PBS.

3. Pass all diluted BM into new falcons through 100 um sieve to remove mini clots.

4. In each of 6 new 50 ml falcons or Leucosep tubes put 15 ml Ficoll and then follow your protocol, ie layer BM, centrifugation, buffy coat ...