I am culturing iPSC-derived as well as primary rat neonatal and adult microglia on collagen IV-coated TC ware. I wanted to perform some flow cytometry experiments and tried detaching cells from the surface with Accutase (40 min incubation at 37C, as described in Reich et al https://www.frontiersin.org/articles/10.3389/fimmu.2020.617860/full#h3), however microglia are still stuck to the surface and even quite aggressive tapping of the well plate does not facilitate their detachment.
Has anybody else faced the same issue and could advise on how to detach microglia from the surface?
Thank you in advance!
A cell scrapper could lend you a hand. Once in a tube, you could shake your cells to give them extra-mechanical strain to force detachment from clusters of cells. Pellet the cells, remove most of the supernatant, leave a very small volume behind. Close the tube and flick the tube to detach cells. This is extra mechanical disruption.
Thanks for your answer Oscar L. Sierra !
I tried 20 min incubation with Accutase followed by mechanical cell lifting. Although I see fewer cells attached after these procedures, the viability of cells is incredibly low.
For now, the only option seems to be plating 3-5 times more cells to compensate for low recovery after detachment.
Aleksandr,
I had similar problem with mouse microglia. I tried several things - also coating with poly-HEMA. It does not work. The microglia cells will find their way to fix firmly to the plastic anyway. They don't care if the plate is coated or not. They do what they want :o)
The best way is to culture microglia on astrocytes (if isolated from animal brain). Then after 2-3 incubations with trypLE, hittin' petri dish and dynamic pipetting you will get satisfactory number of microglia cells.
If you have a microglia monoculture, I would try 1-2 enzymatic (trypLE / acutase) treatment, hitting the petri dish strongly and dynamic pippeting (but not too hard not to injure the cells). There is a lot of effort to detach them always.
Good luck!
Hey, Aleksandr,
So, I'm starting to work with microglia now. It's said that human iPSC-derived microglia needs to be handle with hydrophobic plates and pipette tips. To collect the cells, using enzymatic treatment will be very harmful and can even kill them (as you may have noticed from your viability numbers). A colleague told me that she putted the plate on ice or in the fridge for 10min before collecting the cells - the cold makes them detach. I have not tried this yet, but let me know if it works! Good luck!
We spent WEEKS trying to troubleshoot this exact problem. We were using human microglial cells clone 3 (hMC3) and tested various enzymatic and non-enzymatic solutions as well as tissue-treated and non-tissue treated plates. The enzymatic solutions (Trypsin-EDTA, Accutase, TriplE Selection) worked well for detachment within a few (