I have tried isolating PBMCs (looking to culture monocytes and differentiate into macrophages) from human blood using Ficoll gradient. I get a good amount of cells that attach. After 2-3 hours I wash with PBS to remove floating cells and the attached cells look good, but the next day the majority of them are floating. I am using RPMI 1640 w/glutamine + 1% pen/strep + 10% FBS. I have not yet tried adding other factors like m-csf, but that will likely be my next step.
Just curious if anyone has any input since I've read protocols that FBS should be sufficient for at least culturing the cells for 2 or so weeks. Article Culture and Differentiation of Monocyte Derived Macrophages ...
You could use PBS + 2% FBS during the isolation to avoid stressing out the cells too much.
In addition to your above medium, add sodium pyruvate and NEAA, it will help your PBMC to grow for longer time period.
Just wondering why you wash the PBMCs once plated. Is it to remove non adherent cells and to isolate monocytes that you want to culture further? If you are indeed looking to culture whole PBMCs, avoid the washing step. Majority of the PBMCs are B cells and T cells and these are non-adherent. Therefore, you would expect them to float. Floating does not always mean cells are dead. You could take a small aliquot; stain them with trypan blue to check if your cells are dead or just floating. If they are indeed dead, try optimizing your PBMC isolation/culture to improve cell viability.
If you are indeed looking to culture monocytes, the washing step makes sense. People use it as a crude method to isolate monocytes. Not all cells that stick are monocytes and not all monocytes stick. Therefore, when you wash after 2-3 hours, you may have a significant number of non-monocytes sticking to the plate and these might be coming off the next day. In addition, monocytes constitute
Karsten Krey I did try including FBS once during isolation, but I don't recall the percentage. I will give the 2% a shot. Thank you!
Waqas Nawaz thank you for the advice. I will try adding these reagents to the medium.
Julie Joseph this is really helpful, thank you. You are correct, I am trying to culture monocytes (should have made that more clear in my post title). I have been checking viability by trypan blue prior to plating, but you make an excellent point to check the floating cells the next day after plating. I plan to add M-CSF this next round and see if that improves the outcome. I also read that washing the non-adherent cells off a little sooner helped other individuals.
Thank you all!
Hi Julie Joseph thanks for the detailed answer. I had similar issues when I tried isolating monocytes from PBMCs . In my case very few cells attached to plastic after 2-3 hrs of isolation. I am wondering if longer incubation will increase no. of attached cells? Also If I add MCSF say very next day how long do they take to differentiate into macrophages. As I know its not possible to culture expand primary cells or even freeze then I must use these primary macrophages for experiment straightaway . ?
Thanks
Hi sara ,
From my experience with isolation of bone marrow derived monocytes (isolated from mice ) . I use DMEM complete Medium ( 10% FBS , 1% penicillin/streptomycin). you can grow them at low attachment 6 well playtes from sigma aldrich . Also , monocytes tend to grow in DMEM better than RPMI . you can also increase the percentage of serum instead use 20% FBS . MCSF is important from day zero isolation .
Hope this answer could help !
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