I am working with the THP-1 cell line, and they were differentiated into macrophage-like cells using 5ng/ml PMA. When I try to detach them using accutase (20min-37C), they are successfully detached but most of them die (checked them by trypan blue, or FAM poly caspase (aka FLICA) assay).
Is there any gentler treatment to detach them without affecting their viability?
Gently scraping, could be not so invasive method for detaching of the cells as chemicals. Using of chemicals inhibiting their adhesion could affect the cells viability!
That is come on minde at first shot!
Did someone else have tried another method? I am interested by this question also (Thaks for the question Alsad)!
Good luck!
Thanks Oskan! that's what I was going to try next. As you said, I believe it is a gentler method, and no chemicals involved. People in my lab have successfully detached cells with physical scraping on different type of cells, but, not on THP-1. I will let you know how it goes.
I work with J774.A1 macrophages and scraping is very easy and gently method to detach them. After scraping just wash the flask with culture medium in order to collect the cells. After that you could count them and seed or watever you want to do!
If I could help you, do not hesitate to ask me!
Luck!
If available, you can incubate the cells with a dilute lidocaine solution. That method was used successfully by a colleague who cultured primary astroglial cells, so it shouldn't result in much loss in terms of viability.
Another thought, we have had better luck detaching cells by using sterile plates that are not treated for cell culture and instead treat the plates with a brief incubation with gelatin (rinse this with PBS before seeding the plates). Good Luck!
Which is better for preliminary experiments using ready macrophage cell lines such as J774.A1 or or bone marrow derived macrophages isolated from mice ? is methods of detaching are different in both cases? thanks guys for your time
Thanks Rodrigo and Susan for your input. It is really appreciated!
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