Assay Report Card

ID: ALA3706023

Type: Functional

Description: FLIPR Assay: Compounds were tested for their ability to inhibit C5a-mediated calcium mobilization using a stable cell line over expressing the human C5aR & the Galpha 15 protein on the FLIPR (Fluorescent Imaging Plate Reader) Tetra system. Cells were maintained in culture media containing DMEM (Dulbecco's Modified Eagle Medium) with 4.5 g/L glucose, 10% fetal bovine serum, 100 U/mL, 1x non-essential amino acid, and 250 ug/mL G418 (Geneticin). Prior to testing of compounds, cells were plated at 10,000 cells/well in clear bottom 384-well black plate, and incubated overnight at 37C. with 5% CO2. On the day of experiment, culture media was removed, and replaced with assay buffer HBSS (Hanks' Balanced Salt Solution) with 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 0.1% BSA (bovine serum albumin) BSA containing calcium 5 dye (Molecular Devices) and 2.5 mM probenecid. After 1 hour incubation at 37C., 5% CO2 for 60 min, cells were pre-incubated with compounds for 30 minutes.