Hi, I am planning to isolate single cells from human lung tumour tissue. I am wondering what is the best way to enzymatically degrade the tissue to isolate MOST of the cells (since the population of interest is very rare and I want to avoid false negatives when I do FACS analysis). I came across multiple collagenase cocktails which have been reported in literature, but not sure which is the best way forward. Which is the best collagenase to work with?
Any leads would be appreciated. I don't want to optimise too much since these are precious patient samples. Thank you! :)
You can digest tissue in RPMI-1640 (2-3% serum) containing 0.8mg/ml Dispase and 0.2mg/ml Collagenase P (Roche). Tubes should be incubated at 37˚C in a water bath or incubator and gently invert at 5-10 min intervals to ensure the
contents are well-mixed. After 30 min,gently collect the cells in another tube and add the fresh media with collagenase and dispase. Total process will take around 60 min. Centrifuge the cells and plate them in fresh media.
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