Hi all, when I isolate the immune cells from the lamina propria, after the first media digestion (RPMI 1640, 5mM EDTA, 20mM HEPES, for 20 minutes, 37 C ), I found that some intestine becomes white, and we can't get immune cells from those intestine, Does anybody know the reason, and any strategy to deal with this situation? Thank you so much.
I usually use 1640 with 2 mM EDTA and 5% FBS for the first step of digestion, and 0.5 mg/ml Collagenase IV with 5% FBS for the second round. In the following steps I only add 1 mM EDTA to prevent cell aggregation. High EDTA concentration will cause cell damage.
Definitely try the 5 or 10% FBS first because any incubation step of digesting cells you extract from mice you killed should have FBS as a digestion solution component. Otherwise without serum, you can easily lose like a large portion of viable cells (anywhere from like 20% to over 50%) in your sample. Well they'll still be there but they'll be dead.
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