| Assay Method Information | |
| | In-Vitro CRAC Channel Inhibition Assay in Jurkat Cells |
| Description: | Inhibition of CRAC channels was determined following thapsigargin (Sigma, Cat #T9033) induced endoplasmic calcium release in Jurkat cells, (see Yasurio Yonetoky et. al Bio. & Med Chem. 14 (2006) 4750-4760). Cells were centrifuged and re-suspended in equal volumes ° f Ca2+ and Mg2+ free Hanks buffer and Fluo-8 NW dye (ABD Bioquest, Inc., Sunnyvale, Calif.) loading solution at 2×105 cells/100 μl/well in 96-well black plate. Plate is incubated at 37° C./5% CO2 for 30 min followed by further 15 min incubation at room temperature. Test compounds (DMSO stocks diluted in Ca2+ and Mg2+ free Hanks buffer) at desired concentrations were added to the wells and incubated for 15 min. Thapsigargin (1 μM final concentration) was added to the wells and incubated for 15 min to inhibit the Sarco-endoplasmic reticulum Ca2+ ATPase pump thereby depleting endoplasmic calcium and raising cytosolic calcium concentrations. Store-operated calcium entry was initiated by adding extracellular Ca2+ to a final concentration of 1.8 mM. Fluorescence was monitored over 5 min on a plate reader (BMG Labtech., Germany) with excitation at 485 nm and; an emission wavelength at 520 nm. Data were analyzed using GraphPad Prism. IC50 for each compound was determined based on the percent inhibition of thapsigargin-induced calcium influx into cells. |
| Affinity data for this assay | |
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