I need to purify monocytes isolated from human peripheral blood using the plate adhesion methodology. However, I only have DMEM in the laboratory. I did a pilot and the cells did not adhere appropriately. Has anyone done or seen any protocol using this culture medium or is only possible with RPMI.
Dear Fabricia!
https://www.researchgate.net/post/Are-human-B-and-T-cells-adherent-or-suspension-cells-in-culture-I-think-theyre-sticking-to-the-bottom-of-my-plate-Possible
Anirudh Gangadhar added an answer August 24
Hi Tiffany Guess I used Ficoll to obtain human PBMCs containing lymphocytes and monocytes and I've tried to separate the lymphocytes by suspending the PBMCs on a plate in the presence of DMEM media (serum-free). Monocytes will adhere to the plate surface so you can easily remove the non-adherent lymphocytes. Interestingly, when I stain the adhered cells with CD3+CD16+CD19, I observed a lot of B cells (CD19) but no T cells. I do know that you would lose some lymphocytes in this process but so far I don't have an explanation as to why I'm only seeing B cells.
Hi Fabrícia Lima Fontes
which protocol do you use?
We are using IMDM which is a little more enriched, but i think DMEM should also work, especially for the adhesion part.
Things you should consider:
- Do you add a protein source (HSA, BSA, FCS, Plasma etc?) - Too much Protein may inhibit adherence
- Do you work with fresh samples or cryo-preserved samples? - Cryopreserved samples might take longer to adhere
- What s your adherence time? Too little time might result in incomplete adherence
- Did you have bad luck? Some donor samples simply don't adhere
Some insights in your protocol might help to find a solution
best regards
Robert
Hello Fabricia
I agree with Robert. You can use DMEM but with high glucose content and 5%FBS. Cells may not adhere 100% depending on many factors.
All the best!
- Badges
- Science method
- Similar topics
- Methodology