How to detach adherent monocytes? - Trypsin doesn’t work (Oct/11/2005 )

Hi everyone,
Recently I posted here my question regarding isolation of CD14+ cells from the peripheral blood. I was looking for some cheap and fast protocol. Thegradstudent and roucky recommended adherence of PBMCs to plastic for 2 hours at 37*C. I am doing that and it works – too well. Yes, I have a problem do detach the cells by Trypsin to count them.
Any suggestion? Does it depend on the type of plastic how strong the attachment is? I am clueless…

Thanks a lot in advance.

Paja

Try adding EDTA to the mix.

i don't think your cells will come off with trypsin however much you use, you need to physically scrape them off with special scrapers you can buy. i've just started a new project and this is what i've been doing! it works pretty well.

hi paja. yes, scraping the cells does damage them more than using trypsin, but you can still culture them really well and carry out subsequent studies on them. many of the cells survive this treatment. if you can't use trypsin you may have to resort to scraping. i'm quite new to working with monocytes so i don't know if there are any other solutions to your problem, the edta suggestion might work but i'm surprised my supervisor doesn't know about it if so.

we study adherent keratinocytes; to uplift the cells for either subculture, count, assay, or harvest, we use this stuff:

https://catalog.invitrogen.com/index.cfm?fu...tDescription=50

this is a trypsin-edta solution that works in our cell line. The edta is necessary for uplifting. these cells are strongly adherent.

I know that trypsin-edta solutions come in varying amounts of trypsin and edta in the formula, perhaps if you called Invitrogen's Gibco people and asked your question they could make a recommendation?

good luck