http://gallilab.stanford.edu/protocols/documents/1BMCMCculture.pdf
I've been following the protocol above, but in short:
I extracted bone marrow from mice, washed the cells, and resuspended in 10ml DMEM with 10% FBS, L-glutamine, and pen/strep. I then added 10ng/ml recombinant murine IL-3. 2 days after this, I aspirated the cells, leaving the adherent cells behind, spun them down, and resuspended in fresh media with fresh IL-3. I've continued passaging the cells every 4 days for ~2 weeks now, and my cell count keeps going down, and purity (as measured by c-kit/FCeR1a +/+ count) is ~5%. Does anyone have any tips for what I may be doing wrong?
Your method seems OK but you may extend up to 3weeks..since bone marrow derived mast cells are mucosal type. You may check the characteristics of mast cells by using Alcian blue staining instead of toluidine blue.
Hello
I agree with Rashmi Singh , Kindly see the protocol & suggestions in the link
Good Luck
http://bio-protocol.org/e1053
Hi,
For BMMCs you need to use RPMI or IMDM, this cells are in suspension they are not adherent. In the case of IMDM you need to supplemented with 10 or 15% FCS, 1% nonesenssial animo acids, 1 mM sodium piruvate, 50 μM 2-ME, 100 U/ml penicillin, 100 U/ml streptomycin, and 10 ng/ml IL-3. After 3 or 4 days you have to change the medium, do this until the culture have 4 weeks. At this time you will have madure MC (around 96% c-kit and Fcepsilon positive cells). Try to keep not more then 1.5 millions of cells per mL of medium. In the begening I sugget to resuspend the bone marrow cells in 20 mL of medium, after one or two weeks, you need to star counting the cells and keep the proportion that I just suggest.
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