I used Gibco RPMI 1640 ( 4.5g/L D-Glucose, 2.383 g/L Hepes Buffer, L-Glutamine, 1.5g/L Sodium Bicarbonate, 110mg/L Sodium Pyruvate) medium supplemented with 2% Human serum for monocytes plastic adherence and then i used for macrophages culture the same medium composition plus 50ng/ml GM-CSF to generate M1 macrophages and 50 ng/ml M-CSFfor M2 macrophages.
The problem i have , is that i don't get enough macrophages after 5 or 7 days differenciation and the M1 macrophages are CD206 positve and low CD 86 positive whereas the M2 macrophages are CD206 positive and CD86 positve.
If someone has a good Media composition please share with me
Thank you Juan for your answer, but what is the purpose to add insulin, please tell me more
Dear Gaetan,
I have successfully differentiated and polarised macrophages from human monocytes for the past two years and what I can tell you is:
- use complete RPMI (so the classic recipe, which means +10% FBS - worth trying a couple of batches of FBS when starting the culture and assess which one works better)
- isolate the monocytes via positive or negative isolation (magnetic separation) in order to keep a high level of purity
- pivotal is to plate enough monocytes/mL in order to obtain the so-called "macrophage lawn" after 7 days. You only reach this if you plate at least 1,000,000 cells/mL. If I were you I would plate more towards 2,000,000 cells/mL. Change only HALF of the medium every few days since they are happier when they still have some self conditioned media!
- in terms of CD206 and CD86 expression: M1 macrophage DO express CD206, it is widely reported in the literature so I advise you to read/study the literature a bit more on this. As regard CD86 low: CD86 is induced by IFNgamma so unless you add it to the culture (normally added with LPS if you want to have M1 macrophages), your levels of CD86 will remain low.
Do you add IFNg/LPS for M1 and IL-4 or IL-13 for M2 (depending which M2 subset you are aiming at) at all?
I hope this helps.
Good luck!
Giulia
Insulin, isolated, first, then Insulin and Iron; Finally, catalytic Iron
Agree with Giulia about the media and don't forget to activate them with LPS/IFNg (M1) and IL-4/IL-13 (M2). About the markers , CD209 works quite well for M2 and CD80/CD86 for M2.