were washed by centrifuging at 1500 u00d7 g for 10 minutes at room temperature the platelets were washed with platelet buffer comprising hank 39 s buffer ca free with 0 2 bovine serum albumin bsa and 20 mm hepes the platelet samples were then adjusted to 1 5 u00d7 10 7 ml after which 50 u03bcl of calcium ionophore a23187 together with a calcium chloride solution were added to 50 u03bcl of test drug compound dilution in plates to produce final concentrations of 1 7 u03bcm a23187 and 1 26 mm ca then 100 u03bcl of canine washed platelets were added and the samples were incubated at 37 u00b0 c for 15 minutes after which the reaction was stopped by adding 20 u03bcl of 77 mm edta the plates were then centrifuged at 2000 u00d7 g for 10 minutes at 4 u00b0 c after which 50 u03bcof supernatant was assayed for thromboxane b 2 txb 2 by enzyme immunoassay eia the pg ml of txb 2 was calculated from the standard line included on each plate from which it was possible to calculate the percent inhibition of cox 1 and the ic 50 values for the test drug compounds a canine histocytoma macrophage like cell line from the american type culture collection designated as dh82 was used in setting up the protocol for evaluating the cox 2 inhibition activity of various test drug compounds there was added to flasks of these cells 10 u03bcg ml of lps after which the flask cultures were incubated overnight the same test drug compound dilutions as described above for the cox 1 protocol were used.