a neubauer cell chamber and the cells are adjusted to a concentration of 4 8 u00d7 10 6 cells ml in cell culture medium rpmi1640 supplemented with 10 fetal bovine serum alternatively whole blood containing an anti coagulant is used directly in the assay 100 u03bcl of cell suspension or whole blood is placed in each well of a 96 well cell culture plate then 50 u03bcl of the 4 u00d7 inhibitor stock to the cells is added finally 50 u03bcl of a lipopolysaccharide lps working stock solution 16 ng ml in cell culture medium is added to give a final concentration of 4 ng ml lps in the assay the total assay volume of the vehicle control is also adjusted to 200 u03bcl by adding 50 u03bcl cell culture medium the pbmc cells or whole blood are then incubated overnight for 12 u2013 15 hours at 37 u00b0 c 5 co2 in a humidified atmosphere the next day the cells are mixed on a shaker for 3 u2013 5 minutes before centrifugation at 500 u00d7 g for 5 minutes cell culture supernatants are harvested and analyzed by elisa for levels of il 1b r amp d systems quantikine kits dbl50 tnf u03b1 biosource khc3012 il 6 endogen.