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dear all
i'm about to carry out some incubations using
cryopreserved human and mouse hepatocytes for the first
time. i've managed to find plenty information on thawing
and purification steps but would very much appreciate some
advice on incubation conditions,
eg. incubation volume, cell concentration, resuspension
medium - do i have to supplement krebs-henseleit buffer for
an incubation that is less than 2hr, if so what with? is
another buffer more suitable?
any suggestions welcome
many thanks in advance
Nicola F Smith
Cancer Research UK Centre for Cancer Therapeutics
The Institute of Cancer Research
E Block
15 Cotswold Road
Belmont
Sutton
Surrey
SM2 5NG
nfsmith.aaa.icr.ac.uk
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incubation volume = 0.5 ml
cell concentration = 2 * 10E6 cells/ml (or less)
The commercial Krebs-henseleit buffer we use contains
glucose, but I don't think it is required for <
2hr.
You could add HEPES buffer (10mM) to help control pH.
We also use 48 well cell culture plates with shaking
(under 5% CO2).
I don't have a reference nearby... just about every
issue of Drug Metabolism and Disposition has an
Hepatocyte paper.
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)