Isolation of intestinal epithelial cells - Isolation of intestinal epithelial cells (Apr/30/2010 )
Hi, I was wondering whether anyone could share a detailed protocol on how to isolate viable,intact colonic epithelial cells from mice.
I have tried numerous methods with EDTA where in the colon is cut longitudinally and then incubated in 5-30mM EDTA shaking at 37 degrees for 15 minute shakes.
I've also tried to evert the colon onto a glass rod and then shake in EDTA.
The fractions that come off do not seem to yield any IECs or if they do, they are all dead (by Trypan blue stain)
If there is someone out there who has isolated IECs, I would be immensely grateful if you could provide me with a detailed protocol.
Desperately yours,
Retrograde
retrograde on Apr 30 2010, 12:08 PM said:
I have tried numerous methods with EDTA where in the colon is cut longitudinally and then incubated in 5-30mM EDTA shaking at 37 degrees for 15 minute shakes.
I've also tried to evert the colon onto a glass rod and then shake in EDTA.
The fractions that come off do not seem to yield any IECs or if they do, they are all dead (by Trypan blue stain)
If there is someone out there who has isolated IECs, I would be immensely grateful if you could provide me with a detailed protocol.
Desperately yours,
Retrograde
donīt you use a buffer with cell compatible osmorlarity, at least PBS? EDTA and shaking should give colonic crypts and only a few single cells; incubation of crypts with proteases such as collagenase or pronase will give single cells
donīt you use a buffer with cell compatible osmorlarity, at least PBS? EDTA and shaking should give colonic crypts and only a few single cells; incubation of crypts with proteases such as collagenase or pronase will give single cells