Hi all,
I work with the MC38 colorectal cancer model and the goal is to dissociate tumor infiltrating immune cells with minimal loss. There is also massive tumor cell death if a therapy is working.
I find that the tumor itself is usually covered in mucus when harvested. I use 1mg/ml collagenase and 0.02mg/ml DNase, 37 degrees, 220 RPM for digestion. After digestion, there usually are clumps forming, which decreases the amount cells recovered.
My FACS buffer formula is 2%FBS+2mM EDTA.
Is there anything to optimize in the digestion and resuspension protocol to reduce clump formation?
I use a more extended protocol where the epithelial layer is removed first before digesting the remaining tissue, it could be worth a try.
1. Once the tissue is cut into small pieces, wash 1x with ice cold HBSS
2. Incubate in pre-warmed HBSS+5mM EDTA+1mM DTT for 20mins at 37C whilst shaking (to remove epithelial layer)
3. Wash 1x HBSS
4. Incubate in DMEM (no FBS)+1mg/ml Collagenase D + 0.1mg/ml Dispase II
5. Incubate 30-40mins at 37C
6. Wash HBSS
7. Strain through 70um strainer
8. Perform red blood cell lysis
9. Resuspend in FACS buffer
I have never had a problem with cell clumping and the mucus layer is removed.
Good luck!
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