Isolation of cytotrophoblasts - (Aug/24/2010 )
i have to isolate cytotrophoblasts from first, second and third trimester villi. I am trying to standardize this protocol but i am facing some problems with it
i have tried kliman et al 1986 method and Fisher et al 1989 method . earlier i was trying on frozen samples since most of my samples are frozen, but there was no cell pellet as verified by H and E staining then i shifted to fresh samples now i am getting clear cell pellet but unfortunately there is no separation of cells on percoll gradient, no middle band is formed which is specific for mononuclear cells. i have tried 70-5 % discontinuous percoll gradient ( centrifuged at 2000 rpm for 25 min). percoll gradient is especially important for enrichment of cytotrophoblast, since i need pure cytotrophoblast population, so this step is very important in my work.
please suggest me some changes to sort out this problem in fresh samples and if there is some protocol for isolation of cytotrophoblasts from frozen placental samples.
I don't know about the protocol, but you definietely won't get viable cells out of frozen tissue.