I am trying to prepare mononuclear cells from mouse colon lamina propria. I tried either with classical enzymatic digestion (by removing epithelial cells with EDTA and shaking, and then digesting the mucosa with Liberase) and I also tested the Gentlemacs dissociator from MiltenyiBiotec (this one is for the small intestine and does not seem to work perfectly with colon in my hands). The viability of the cells at the end of the protocol is rather low. Usually I perform a percoll separation at the end of the protocol to get rid of remaining epithelial cells and dead cells. However the cells die in the following hours, I'm unable to keep them alive in complete medium (RPMI 10%FCS 2mM Glutamax 50µMbetamercaptoetOH). If someone could help me with technical advice or a protocol, it would be great.
Hi,
Your protocol seems okay to me, but I'm not sure that mononuclear cells can survive long in complete RPMI medium. If I remember corectly, we used mononuclear cells immediately efter extraction, we never grew them after.
Perhaps you should try to extract mononuclear cells fom mouse blood first, to check that your medium is the optimal one for them.
Then, if everything is good, try to soften the extraction conditions.
I'm sorry, but I can't provide you help with this last point.
Hi, Maybe you can try this protocol which I used before when I was working on the LP cells.
I really need to keep them for e few hour/days so maybe i will try to modify the culture medium. Thanks for your advice!
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