Handling Fresh Hepatocytes in Suspension
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实验试剂
Dexamethasone, 10 mM in DMSO solution, 1 x 250 μL (supplied in excess)
Maintenance Cocktail solution, 1 x 20 mL, containing:
2.5 mL penicillin-streptomycin, 10,000 U/mL
5.0 mL ITS (insulin, transferrin, selenium complex, BSA, linoleic acid)
实验步骤
1. Centrifuge and resuspend hepatocytes
1) Remove tube(s) of hepatocytes from shipping container; record lot number.
2) Slowly invert tube a few times to mix cell suspension.
3) Centrifuge hepatocytes at 4°C :
Human hepatocytes - 100 x g for 10 min
Animal hepatocytes - 76 x g for 6 min
4) Aspirate supernatant carefully, leaving approximately 1 mL of medium in tube.
2. Determine viability and viable cell yield by Trypan Blue exclusion count
8) Determine viability of hepatocytes (live cells/total cells x 100).