| Assay Method Information | |
| | Reverse Mode Assay |
| Description: | The assay is based on the monitoring of intracellular Ca2+concentrations using the calcium-sensitive dye Fluo-4. CHO cells expressing NCX1 were loaded with the dye by means of the acetoxymethyl ester Fluo-4 AM (Invitrogen, F14202), which is cleaved intracellularly by esterase activity to yield the charged species of free Fluo-4. After an preincubation period with the test compound, Gramicidine (Sigma, G5002) was added. Gramicidine is an ionophor for Na+ions mediating an increase of intracellular Na+ions. Consequently, intracellular Na+ions are exchanged against extracellular Ca2+ions (Ca2+influx, reverse mode). The intracellular elevation of Ca2+ ions was detected by measuring the fluorescence of Fluo-4 at a wavelength of 520 nm by a FLIPR device.Briefly, for the reverse mode transport assay 18000 cells per well were seeded into a 96 well microplate (Corning COSTAR 3904) and incubated overnight in culture medium (1x Nut Mix F12 (Ham) (Gibco, 21765-029); 10% (v/v) fetal calf serum. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |