Isolation of lymphocytes from mouse stomach. Major help needed. - (Jan/08/2009 )
Does anyone have a protocol or an idea on how to isolate lymphocytes from a mouse stomach? There is no set protocol in the lab and
I need to isolate lymphocytes for flow cytometry. I have tried to make up my own protocol however the sample was extremely dirty and I was not able to detect any lymphocyte population. If anyone can give me any ideas I would greatly appreciate it.
-Lost and confused
I have never done such a isolation... and worse I am not specialist in the domain of stomach
My idea is to clamp the stomach before the duodenum, recover the organ and wash the outside in PBS for example
then wash the inside by injecting some buffered solution through the other end, mix well and throw out; many times certainly...
I would make now incision all along the organ to obtain a flat material; make some little pieces and let a typical trypsin-free cell dissociation solution (Sigma) working for some minutes; I would mash the remaining pile on cell strainers...
centrifuge, wash and first try a positive selection (MACS) with anti-CD3-coated magnetic beads... If it does not work, put in culture with some cytokines, but in this case a biased selection could appear
Thank you for the reply. I will seeif this works.
Wouldn't it be easier to isolate from the Thymus and Spleen?
I suppose sciV85 want to characterize the gatric mucosal population...
Yes. I need to evaluate the lymphocyte population in the stomach since the bacteria I study directly infects the stomach.