Hi, I am working with macrophage-cancer cell co-cultures. I have been trying to detach THP-1 differentiated macrophages (grown in RPMI-5% FBS) from tissue culture plates for FACS analysis. But I have been unsuccessful, as I would have 30-40% dead cells. I have used cell stripper solution (10 min at RT) and PBS with 5mM EDTA (10-15 min on ice) with multiple pipetting rounds post incubation. But neither has worked. Is there any plate preference? I would highly appreciate if someone can advice me on how to overcome this issue.
Thanking in advance
You can try using plates which are not cell culture treated. Macrophages normaly adhere less strongly on them. When using PBS/EDTA on ice you have to wash several times to remove all traces of medium.
Have you tried just scraping them? They do not attach very strongly and scraping may get many more up without killing them. Just a thought. A petri dish as Birgit Vogt suggested may also be an option. Good luck!
Try to use cold accutase solution at 4c for 8 min or put the plat at cold room for ten min then use Rpmi with pipeting a few time to mske the cell detached easily with high rrate of life cells
You could also try Accutase - this is slightly different than standard trypsin-EDTA in that it is combination of proteolytic and collagenolytic enzymes that ensures that surface proteins and epitopes stay entirely intact. I also find a slightly greater viability among primary cells (I have used it for PMA activated peritoneal macrophages) compared to Trypsin-EDTA.
Birgit Vogt I am differentiating them currently on non treated plates. Lets see how that goes. Thank you !
Michael Davis They do attach very strongly to plates. hence scraping didn't work well for us.
Nawzat Aboziad Issa Thank you for the response
Kuhulika Bhalla Thank you, I have placed an order for accutase. Hoping this works.
I appreciate the help.
First, i treat THP-1 derived macrophages plate with trypsin ( 100mm petridish/ 1ml trypsin) and keep it in incubator for 10 minutes. Then i give medium thrust along the rim of the petridish using palm. This thrusting helps further detaching the cell. then add RPMI media and collect cells.
- Badges
- Science topic
- Similar topics
- Agriculture
- Horticulture
- Seeds