Calcium flux assay

Calcium flux was measured in stable transfected XCR1-expressing HEK293 cells, generously provided by Dr. Joseph Hedrick (Schering-Plough Research Institute) (69), using a previously described method (66). Briefly, cell cultures were grown to ~90% confluency, lifted from the culture plate, washed 2x in warmed PBS, and suspended in assay buffer (1x HBSS, 20 mM HEPES (pH 7.4), and 0.1% BSA). Cells were plated at 2.0 × 10⁵ cells/well in 100 μL in a 96-well plate and incubated with 100 μL of FLIPR Calcium 4 Assay Dye (Molecular Devices) for 1 h at 37 °C and 5% CO₂. A Flexstation 3 (Molecular Devices) was used to treat cells with various concentrations of purified proteins and monitor calcium flux. All experiments were conducted in triplicate.