Hello, everyone! I got a problem in harvesting enough bone marrow derived macrophages (BMDMs). I used both legs of one mouse, and inoculated bone marrow cells to two 75 cm^2 flasks without filtering them through 70/40 μm strianer or lysing red blood cells (I intended to lower these kinds of stimuli to cells). In the Day 7, I got only 1-3 x 10^6 cells totally despite using 10 ng/mL reM-CSF (Prospec). What was the problem about my procedure? I would very appreciate it if anyone could give me help?
The problem may be the period of lysis.10-30s would be enough. Actually, you dont even have to do the lysis.
When you look at your flasks on day 7 do you see many cells? You only mentioned that you have a low cell count. I ask because macrophages are notoriously 'sticky'. It is possible that you are not detaching many of the cells from your culture dish (don't use tissue culture treated plastic. Or the cells could be clumping during the centrifugation step giving you a lower yield.
I started a thread about the best ways to dissociate macrophages a few months ago. https://www.researchgate.net/post/How_do_I_prevent_the_clumping_of_bone_marrow-derived_macrophages_for_flow_cytometry2?_tpcectx=profile_highlights
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude