In literature, I see various concentrations for GM-CSF and/or IL-4 to differentiate hPBMCs into macrophages or DCs. What are the concentrations that you guys use? And what might be the reason for such a great variation in different protocols? Do I need to optimize the concentrations in my hand based on cell-death, differentiation-efficiency or ...?
In general you first should isolate monocytes from PBMCs. There are several different methods, like magnetic sorting or plastic adhesion to isolate monocytes from human PBMCs.
Afterwards it depends on the source of your cytokines and their activity. To generate DC-SIGN, CD1a positive and CD14 negative DC's I frequently used 500 U/ml of IL-4 and GM-CSF both provided by the company Immunotools. Monocytes were cultured in a density of 1 million cells per ml in a 24 well plate in RPMI 1640 medium with 10% FCS, 1% Gln and 1% Pen/Strep. The Medium containing IL-4 and GM-CSF was renewed after 3 days and on day 6 DCs were generated. Afterwards stimulation with LPS or other PAMPs leads to the maturation of DCs.
To generate Macrophages many people just leave out the Il-4.
You must purify monocytes from PBMC. For macrophage, you can use 50ng/ml GM-CSF and for DC, use 50ng/ml GM-CSF and IL-4, 20ng/ml. You can also see this paper
http://www.ncbi.nlm.nih.gov/pubmed/25870903
Definitely confusing these MQ and DC protocols. I worked with mice, but it seems that the cytokines are just used at higher concentration working with blood-derived monocyte.
With respect to MQ: classically (1970s) MQ were isolated simple by cultivating hPBMC, discarding the suspension cells and continuing with the adhered cells. 2-3 cycles are enough.
With respect to DC, the following 2 protocols are rather consistent even after a decade.
http://www.jimmunol.org/content/170/8/4069.full (2003)
http://www.bio-protocol.org/e1194 (2014)
Coincide in
1. start from isolated monocytes from hPBMC
2. stimulate with 1000U/ml or 100 ng/ml GM-CSF + 500 U/ml or 50 ng/ml IL-4 for 24h - 3d.
3. followed by a 24h pro-inflammatory stimulus: 100 u/ml TNFa, 10 ng/mL IL-1beta, (1 uM PGE2), or other activating stimuli.
4. Most investigators have the experience that MQ adhere strongly whereas DC only at a minimum.
Considering you work with human cells avoid FBS, and if you do heat-inactivate.
My personal experience is that most protocols generate M1-MQ or inflammatory DC, Literature on the generation of M2-MQ or non-inflammatory DC is much more scarce and mainly described for mice.
GOOD LUCK
If you are interested in M2-MQ
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