Hello everyone. I am researching the influence of CD8+ T cells on macrophage polarization in vitro. But I don't know how to co-culture them, e.g. plates, cell concentration, culture time, etc. I think CD8+ T cells should be activated by CD3/CD28 before adding to the co-culture media. Do I also need to stimulate macrophages in advance? Should I use M-CSF or LPS+IFN-γ? What's the difference between macrophages derived from bone marrow and peritoneum? I appreciate it if anybody could give me some advice.
Hi, I think you should activate macrophages to study the cross-talk between MAC and CD8+. For MAC culture you can use DMEM ( you can also use RPMI medium) supplemented with 10%FCS, 1%PSA and M-CSF ( 20ng/mL). On day 2 or 3 ( depending on the morphology of the cells) you can rinse and immerse the culture in fresh medium. ( working with primary cell cultures can be tricky, so to be sure that the cell culture would survive you should let the MACs attach to the culture vessel for more than 24h). Depending on the MAC phenotype that you would like to study you have to activate MAC culture with LPS+INF-γ ( 100 U/mL of INF-γ and 100 ng/mL of LPS) for the M1 phenotype and IL-4 ( 20ng/mL )for the M2 phenotype ( both polarizing states of MAC). For the co-culture, MAC should be grown to confluence on the bottom of the transwell co-culture chamber at a density of 5x104 .
PEMs ( peritoneal macrophages ) and BMDMs ( bone marrow-derived macrophages) are phenotypically distinct and have different expressions of lipid metabolism genes.
For more information on MAC co-culture systems and protocols you may refer to the papers attached below.
I hope you find this usefull! Good luck with your research !
Article The expression of MMP-1 and MMP-9 is up-regulated by smooth ...
Article Comparison of the characteristics of macrophages derived fro...
https://www.nature.com/articles/srep35234
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