I use PMA to differentiate thp-1 and u937 cells to macrophage. I usually use CD11b as differentiation marker but I need another one.
CD14 should be a good marker of differentiation. Upon PMA treatment, the CD14 level goes up to about 60% from around 10%. CD14 is a TLR coreceptor and is present on a small proportion of the undifferentiated cells.
I will prove with the CD14. I don't know about the Non-specific Esterase but I am looking for information. Thanks!
Hello Virginia,
We have tried several models for differentiating monocytes, hopefully these could also be used for cell lines. We found that HLA-DR is particularly useful for PMA as well as for IFN-gamma differentation on normal human monocytes, also CD71. But i would start with HLA-DR. These antibodies are easy to obtain with people working in HLA typing. Best regards
Well, firstly you should address whether THP-1 is becoming a M1 or M2 macrophage.
So, you should stain with CD206 (for M2 subtype) and CD16/32 (M1 subtype). CD14 will stain either macrophage subtypes and is expressed even in undifferentiated human monocytes (upon activation you may see CD14 upregulation).
However, PMA usually is known to activate macrophages into a so-called M0 subtype, where they are not differentiated into macrophages. The activation with PMA in vitro usually takes 72h to happen and the PMA concentration may vary according to experimental conditions (from 20ng/mL to 100ng/mL in some papers).
Hope this helps!!
Kind regards,
Raphael
Hi,
Assuming you have access to a multi-colour flow cytometer and access to the antibodies mentioned by Raphael here. I would recommend you to go for a combination of CD14, CD16 and CD206. The possibility of sub-gating the CD14+population would provide you a detailed answer and might even surprise you (the same stimulant at the same concentration and fixed exposure time might drive these two different cell lines to different [M1/M2] phenotype). In my knowledge, Thp-1 cells tend to take an M1 phenotype more often.
cheers!
Jose