Hi !I gotta a question about Procedure 3(g), how could you eliminate...

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yu liming
Sep 4, 2013
protocol Protocol: Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)
Hi !
I gotta a question about Procedure 3(g), how could you eliminate erythrocyte and fragments of bones? I usually use red cell lysis buffer as well as filter with 200 nylon mesh , but the rate of adhesion is very low . what`s the purpose of incubating FBS at 50 °C for 30 min before
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yu liming Answered Sep 9, 2013

Nanjing Medical University

Thank you very much. Here is my email address:yuxiongbao8@gmail.com I wish we could exchange experience later.
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Ran Chen Author Answered Sep 9, 2013

Zhejiang JFK Biological Technology Co. Ltd.

No. I don't suggest to recycle the BMM growth medium. You can use the L929-conditioned medium (L-cell conditioned medium) instead of M-CSF to lower the cost. Actually, the L929-conditioned medium is used in this protocol.

-- by Ran Chen
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yu liming Answered Sep 7, 2013

Nanjing Medical University

thank you for your suggestions.The culture cells are better than last time.Here is a question: could BMM`phi` growth medium be used for recycle? I worry about the concentration of M-CSF.
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Ran Chen Author Answered Sep 6, 2013

Zhejiang JFK Biological Technology Co. Ltd.

With the protocol, you won't get significant contamination of erythrocytes or bone fragments. You don't need to eliminate the erythrocytes or bone fragments immidiately --- those residual erythrocytes or bone fragments will be washed out when you change the media after the BMM cells are attached.
50oC to inactivate some unknown cell inbitors in the FBS.
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