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pubmed:abstractText |
A rapid, colorimetric, microassay for detection of agents which are known agonists/antagonists of protein kinase C (PKC) was developed, utilizing their effects on adherence of EL-4.IL-2 cells. Cells that were incubated with agents which are known inducers of PKC activation, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), mezerein and indolactam V, readily adhered to wells of 96 well microtiter plates within 1-2 hours, whereas cells incubated with the negative PKC activator, 4 alpha-phorbol 12-myristate 13-acetate (4 alpha-PMA), which is structurally related to PMA (4 beta-PMA), did not adhere. The adherent cells withstood repeated vigorous washings with tissue culture medium. Adherence of EL-4.IL-2 cells in the presence of PMA could be blocked by the addition of two known inhibitors of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride and staurosporine. Detection of the presence of adherent cells was accomplished by the addition of a tetrazolium salt to culture wells and determination of the remaining viable cells by scanning using a multiwell spectrophotometer (ELISA reader). The EL-4.IL-2 adherence assay meets several important criteria for use as a primary screen in the detection of potential PKC agonists/antagonists, i.e. its selectivity, simplicity, rapid performance through automation and reproducibility.
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