Hi, I need help in detaching adherent monocytes from well plates.
I isolated the PBMCs from buffy coat and I did CD14+ positive selection to collect only the monocytes. I let them adhere on my well plates for 1 hour and co-incubated with my pathogen for 24 hours at 37C with 5% CO2. I used serum-free RPMI and Pen/Strep for this.
After 24 hours, I removed the media and washed with room temperature PBS. Then I added pre-heated TrypLe and incubated them at 37C for 20 minutes. After inculbation, I inactivated the TrypLe by adding PBS. The cells were still very adherent after this so I used a cell scraper to harvest the monocytes. When I checked the cell number after harvesting, I did not see enough cells even though I was sure there were so many monocytes before harvesting (as you can see in the photo 1). I centrifuged the cell suspension that I harvested at 1500 rpm for 10 minutes and saw a pellet that floats around and cannot be suspended (see photo 2). I checked this pellet and it contains all my monocytes, it seems they clumped together but I don't know exactly what happened (see photo 3).
If you know the reason could you please tell me? And if you have some experience with monocyte detachment, could you also share to me? Thank youuuu :(
You can put the well-plate which contain the isolated monocytes on top of ice for around 5 to 10 min and then try to shake the cells gently so the monocytes will be detached from the bottom of the plate. Good Luck.
I used human CD14 positive selective monocytes for macrophage differentiation. I used pre-warm TrypLE. But 20 min never worked. I used to leave them in the incubator as long as they did not detach and kept looking them under the microscope. When they started detaching, I pipetted them forcefully. To stop TrypLE action, I used to add RPMI+10% FBS.
- Badges
- Science topic