Hello everyone,
My name is Paul and I need to isolate microsomes from keratiocytes (HaCaT cell line and human primary keratincytes) to perform then CYP assays.
I've been recommanded to use sub cellular fractionnalisation but I've also seen kit (eg ab206995 from abcam) that does not require utracentrifugation but only slow centri (20K g).
Has anyone ever tested this kit?
Or do you have a specific protocol do advise with this kind of cells?
Hello Paul, in this reference you can find the working protocol you are looking for.
Toxicol Lett. 2000 May 19;115(2):165-72.
Cytotoxicity and keratinocyte microsome-mediated mutagenic activation of carcinogens in cultured epidermal cells.
Chun HS1, Kuzmicky PA, Rucoba L, Kado NY, Rice RH.
Author information
Abstract
Four model carcinogens (aflatoxin B(1), 6-nitrochrysene, 3-amino-1-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were examined for their ability to inhibit the growth of cultured human and rat epidermal cells. To find a basis for observed differences in growth inhibition, aflatoxin B(1), Trp-P-1 and Trp-P-2 were tested for activation by microsomes isolated from these cells in a bacterial mutagenesis assay. Treated rat cultures exhibited sensitivity to Trp-P-1 and Trp-P-2 and especially aflatoxin toxicity (growth inhibition) despite their microsomes being unable to induce bacterial mutagenicity. In treated human cultures, the toxicities of Trp-P-1, Trp-P-2 and AFB(1) were stimulated by 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), consistent with their dependence on the biotransformation reactions this agent induces; however, the toxicity correlated poorly with observed bacterial mutagenicity mediated by their isolated microsomes. 6-Nitrochrysene, a known direct-acting mutagen in bacteria, was highly toxic to the rat but not to the human cells. Since toxic effects can modify carcinogenic outcomes, these findings are compatible with a complex relationship between toxicity, mutagenicity and carcinogenicity and indicate the utility of keratinocytes for clarifying this relationship.
PMID:
10802392
[PubMed - indexed for MEDLINE]
thank you very much José.
Disruption with the potter is enougth to homogenize the cell sample?
And have you heard about the kit that use homogenization buffer and slow centrifugation?
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