each well and added to a costar polypropylene 384 well plate using the beckman multimek workstation 5 u03bcl of surefire perk activation buffer is added to each well and mixed by gentle agitation on a plate shaker for 2 minutes 5 u03bcl of activated lysate from each well is then transferred to a 384 well proxiplate perkin elmer catalog 6008280 the reaction mix is prepared from the alphascreen protein a detection kit the anti igg protein a and streptavidin beads are diluted 60 u00d7 in reaction buffer under green light they are extremely light sensitive 6 u03bcl of the working reaction mix is added to each well under green light and the proxiplate is sealed with an aluminum plate seal the plate is shaken for 5 minutes after which it is stored at room temperature for at least 2 hours prior to reading on an alphaquest plate reader perkin elmer the perkin elmer fp 2 campfire assay kit catalog fpa20b040kt is used to determine agonist potencies for s1p1 in whole cells 1 u00d7 camp antibody solution and 1 u00d7 alexa fluor is prepared as described in the campfire assay protocol the test compounds are dissolved in dmso and then diluted to final concentrations about 9 nm to 0 0005 mm in the assay buffer composed of 2 mg ml faf bsa final 1 mg ml 1 mm cacl 2 0 5 mm final 5 mm mgcl 2 2 5 mm final in pbs ten microliters of the test compound dilutions are placed into 384 well assay plates ten microliters of buffer are placed.