I want to check the heterogeneous population of cancer cells in a particular tumor.
Cut tumor to small pieces with blade and then incubate in 3.5 ml of digestion buffer below per 0.2 g tumor at 370C for 2-3 hours with shaking (200 rpm). Pass cells through 40 um cell strainer with 10 ml PBS and centrifuge cells down (5 min at 1000 rpm). Resuspend the cells in medium or buffer for your next assay. If you need to get rid of red blood cells, add 1/5 volume of ammonium chloride ( Stemcell, cat#7850) for 5 min and wash cells with PBS twice to remove ammonium chloride.
digestion buffer (20 ml):
RPMI 20 ml
l-glutamine 200 ul
Penicilline streptomycin 200 ul
Collagenase type I (Sigma # C0130-500MG) 40 mg
1 mg/ml DNase I (Worthington # LS006333) 800 ul
250 mg/ml BSA 10 ul
1M CaCl2 100 ul
Good luck!
Cut tumor to small pieces with blade and then incubate in 3.5 ml of digestion buffer below per 0.2 g tumor at 370C for 2-3 hours with shaking (200 rpm). Pass cells through 40 um cell strainer with 10 ml PBS and centrifuge cells down (5 min at 1000 rpm). Resuspend the cells in medium or buffer for your next assay. If you need to get rid of red blood cells, add 1/5 volume of ammonium chloride ( Stemcell, cat#7850) for 5 min and wash cells with PBS twice to remove ammonium chloride.
digestion buffer (20 ml):
RPMI 20 ml
l-glutamine 200 ul
Penicilline streptomycin 200 ul
Collagenase type I (Sigma # C0130-500MG) 40 mg
1 mg/ml DNase I (Worthington # LS006333) 800 ul
250 mg/ml BSA 10 ul
1M CaCl2 100 ul
Good luck!
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