I am harvesting BM cells from bilateral femurs, tibias, pelvic bones and humeri from WT mice ages 8-12 weeks, followed by c-kit magnetic bead enrichment and then flow cytometry to sort out the LSK cells. The numbers of expected LSK cells I have found in the literature and on this board seem to vary and wanted to get an idea of what to expect.
I would say around 10,000. Around 6000 if you just take hind limb bones.
Dear Nahal
After c-kit magnetic selection you should have about 2x10e6 cells left. From those about 80000 to 100000 would remain after sca selection. As far as I understand these numbers are reduced if you add a real lineage+ depletion step before you enrich for c-kit.
We start from femurs + tibias only, total cell counts:20 - 30 x 10e6.
How many do you get???
Best
Robert
Up to 10,000. Depends on the mouse age and desired purity. If you want to get highly pure population of SKLs you may get around 2,000 cells.
Depends on the method you use to purify the LSK cells. We purify the cells by FACS and we always use 3 mice per purification. Total bone marrow cells from 4 bones per mouse were marked with the antibodies and sorted. With these conditions, we can obtain up to 30000 LSK cells per mouse.
Thank you for your responses! I was getting 3000-5000. I got a better yield with flushing the long bones. However, after increasing the amount of beads for ckit enrichment and switching the Sca-flourophore for sorting, my yield jumped up over 15-fold which made me skeptical of my purity. I was told to expect ~50000 LSK cells from 2-3 month old WT mice using those bones but I did not find much support for that in the literature.
From femur and tibia using the crush method followed by ckit MACS and FACS I get 150,000 FACS sorted LSK from a single mouse.
We crush legs with pelvis, spine and on occasion sternum and use c-Kit AutoMACs and then flow sorting. Roughly get 120K LSKs / mouse
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