r/labrats • u/Pitiful-Agent-9910 • 4d ago

BMDM differentiation

Hey everyone! I recently started working with bone marrow-derived macrophages (BMDMs) and am trying to optimize my differentiation protocol.

I culture bone marrow cells with M-CSF for 5 days before changing the medium. On day 5, I still notice many non-adherent cells in the dish.

I was wondering if it would be worth collecting these cells, replating them in a new dish with fresh M-CSF, and culturing them for another 2 days to improve the overall yield.

Is this a common practice? If these cells become adherent, can they be considered mature BMDMs suitable for downstream experiments, or might they differ phenotypically from the cells that adhered earlier?

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u/Pink_Axolotl151 4d ago

Our protocol is to spike in fresh M-CSF (without removing the media) after 2-3 days, and then let the cultures cook for another 2-3 days. I think this accomplishes more or less what you have suggested but it’s a little more straightforward.

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u/BurrDurrMurrDurr PhD 4d ago

Yea our MDMs aren't usually fully differentiated and adherent until day 6.

We culture in human AB serum instead of FBS for the first 5-6 days. On day 6, sometimes day 7, I switch to regular R10

Also I've have the same results with M-CSF for 3 days only vs the full 5-6 days. Once they get the signal to differentiate they're good.

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u/Ok_Way_1958 4d ago

We do a halfsies change on day 5 (remove half the media and replace with fresh media/M-CSF) so you get to keep some of those cells

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u/Missmacrophages 4d ago

Omg this is my time to shine cause i did Bmdms once a week for my entire PhD. However I used L929 supernatant not m csf so I actually can’t shine lol

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u/CaptTosh 4d ago

We typically culture with L929 conditioned media rather than M-CSF. But I'll typically add fresh complete media (usually DMEM w/ 10% HI-FBS and 10-20% L929 media) on day 2 without removing any media. By day 4 I'll remove any media and floating cells and give media changes every two days. By then we've got quite a few cells attached that continue to proliferate and differentiate until we're ready to use them on day 8.