TCH_DELFIA_cell_fixation

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