2 replies to this topic

[retrograde]

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

Edited by retrograde, 30 April 2010 - 04:09 AM.

[Inmost sun]

retrograde, on Apr 30 2010, 12:08 PM, said:

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

don´t 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

[retrograde]

don´t 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
[/quote]

Are you suggesting that I use collagenase, pronase/ dispase digestion to replace the EDTA washes ? The EDTA is dissolved in PBS without Mg or Ca. Shaking in PBS alone does not yield any cells as the epithelium is still attached to the basement membrane. The reason I want intact crypts and sheets of epithelial cells is because epithelial cells derive survival signals from adjacent neighbouring epithelial cells as well as from below, thus I reasoned that isolating crypts and sheets would retain this signaling and avoid apoptosis. Have you isolated any IECs before ? Could you perhaps hook me up with a detailed protocol ? Thanks for the advice.