Hi,
I am trying to isolate monocyte from PBMC and induce them to make macrophages and microglia. I used monocyte attachment media and then change after 2 hours to media required for macrophage and microglia. However, next day my cells are dying.
Is there any suggestion or additional growth factor which can be added to increase viability.
Thanks
Dear Atoshi Banerjee
I am not 100% sure that PBMCs (which have a completely distinct ontogeny compared to microglia) will ever acquire microglia like features in vitro. To this end I would suggest you rather check out the attached (rather detailed) review on in vitro models for microglia.
Article An Overview of in vitro Methods to Study Microglia
They do propose at least three cell line derived models and also link to literature that provides ESC/iPSC derived microglia.
To derive macrophages from PBMCs please follow the tried and tested protocol that's nicely laid out on the Bowdish lab page. I've always used this one with excellent results.
http://www.bowdish.ca/lab/wp-content/uploads/2014/11/Human-Macrophage-Culture-2.pdf
Hope that helps.
All the best & good luck with your experiments,
Michael
To differentiate the monocytes into macrophages add GM-CSF which will also increase the survival of these cells.
I'm agree with Caroline, but the cytokines you will add are different if you want M1 or M2 macrophages.
M1 : GM-CSF and IFN-g
M2 : M-CSF and IL-4
You can also stimulate your monocytes with LPS.
Concerting Microglia differentiation I don't know how to do.
Thank you all for the replies.
I was wondering if anyone has tried inducing microglia from PBMC as suggested by
1. A novel in vitro human microglia model: Characterization of human monocyte-derived microglia.
2. Direct induction of ramified microglia-like cells from human monocytes: Dynamic microglial dysfunction in Nasu-Hakola disease.
Thanks
Atoshi
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