Cellular DELFIA Assay Protocol Materials: - 96 well, white, clear bottom, tissue culture treated plates (Wallac Isoplate TC # 1450-516 or PerkinElmer ViewPlate # 6005181) - Formaldehyde - Triton x 100 - PBS - HEPES - Bovine Serum Albumin - DELFIA Assay Buffer # 1244-111 - Primary antibody (mouse or rabbit) - DELFIA Europium-labeled secondary antibody (mouse # AD0124 or rabbit # AD0105) - DELFIA Wash Concentrate # 1244-114 - DELFIA Enhancement Solution # 1244-105 Day 1: - plate cells (density depending on cell line; 20 000/well in 96 well plate for CHOK1 cells) - incubate at RT for at least 60 min before placing the plates in the 37ºC CO2incubator Day 2: - change to serum-free medium for overnight cell starving Day 3: - treatment of cells (compounds in culture medium or in PBS 20 mM HEPES) - fixation (4% formaldehyde & 0.1% Triton in PBS) - block (0.1% BSA in PBS) - primary antibody 0.3 – 1µg/mL in DELFIA Assay Buffer at least 2 h - wash (DELFIA Wash Concentrate, automated cell washer or using multichannel pipette) - secondary antibody 0.3µg/mL – 1µg/mL in DELFIA Assay Buffer at least 1 h - wash (DELFIA Wash Concentrate, automated cell washer or using multichannel pipette) - 200µL Enhancement Solution + 5 min shake for Eu and Sm - Measure Europium time-resolved fluorescence