Hi :) I used to isolate MVs from primary microglia washing them (after polarization or stimulation) in PBS+/+ (calcium and magnesium) and then with 1ml of KRH solution composed by:

· NaCl 125 mM

· KCl 5mM

· MgSO4 1.2 mM

· KH2PO4

· CaCl2 2mM

· D-glucosio 6 mM

· HEPES/NaOH 25 mM (PH=7.4)

After that, cells are stimulated for 30' at 37°C with ATP (1mM) dissolved in KRH solution to induce MVs/EVs release.

Thank you very much for your answer, and just want to see if I get it right- you isolate the MVs/EVs after the 30 min. stimulation, right?

Yes! As I told you the cells are stimulated with ATP (1mM, 1: 100) dissolved in KRH to induce the release of the micro-vesicles, and left in the incubator for the next 30 '. The supernatant consisting of KRH + ATP is centrifuged at 4°C 1200 rpm for 5 'and then at 4 ° C 1000 rpm 5', to eliminate any cellular debris, and then again centrifuged at 4°C 10000 g for 30 'for get the micro-vesicles. After the latter centrifuge, the microvesicles settled and are inside the pellet, which is finally resuspended in DMEM -FBS + P / S + AnfB.

Hope everything is clear!

Good luck!

hello! Is this protocol used for primary culture of rat or mouse microglia or cell line? Another question is, do you think that if I isolate adult mouse microglia with CD11b, would ATP stimulation work in suspension? Thanks in advance

Hi Victoria, I used it for cell line of human microglia. If I remember right I added ATP to the medium (didn't use KRH) for 30' and then replaced with fresh medium (I wanted to get rid of the ATP), but I didn't have increased amount of EVs or good yield of EVs anyway, so I didn't continue with it. Maybe if I would follow the protocol exactly maybe it would've worked.

I guess that there is no difference in ATP stimulation in suspension, it just that it is easier to start with supernatant without cells when you isolate EVs.

Good luck!

Hi Orit, thank for your answer.