| Assay Method Information | |
| | YFP-Halide Influx Assay |
| Description: | YFP-Halide Influx Assay for the CFTR-ΔF508 Mutation and Suppressor mutants (I539T or G550E)The YFP halide influx assay measures the functionality of the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) channels in the cystic fibrosis bronchial epithelium cell line CFBE4lo−. The fluorescence of the yellow fluorescent protein (YFP) variant YFP H148Q, I152L or variant YFP H148Q, I152L & F47L is substantially quenched by iodine, a halide that is efficiently transported by CFTR. The assay is thus used to evaluate the effect of corrector compounds on CFTR channel function by measuring the extent of YFP signal quenching. (Galietta et al. American Journal of Physiology Cell Physiology Vol. 281 no. 5, C1734-C1742, 2001; Nagai et al., Nat Biotechnol. 2002 January; 20(1):87-90.)For this purpose, HEK293 cells are transfected with plasmid DNA containing F508del CFTR, F508del/I539T CFTR or F508del/G550E CFTR and seeded in 96 well plates (70,000 HEK cells/well). The next day, cells are treated with test compounds.Cells are treated with test compounds for 24 h at 37° C. to allow trafficking of corrected CFTR to the membrane.The next day the CFTR channels are activated by treatment with the cAMP inducer forskolin (10.67 μM) and potentiator GLPG1837 (0.5 μM) in 1×D-PBS (from Gibco, Cat n#14090-091) for 20 minutes prior to addition of an I− solution (137 mM NaI, 2.7 mM KI, 1.76 mM KH2PO4, 10.1 mM Na2HPO4, 5 mM glucose). The I− induced quenching of fluorescence is recorded immediately after injection of for 7 seconds. The capacity of a compound to increase number of channels, and therefore overall halide influx is directly correlated with the decrease in fluorescence, and is expressed as (1−(fluorescence after 7 seconds (F)/fluorescence before injection (F0))) and an EC50 can be derived from a (1-F/F0) vs compound concentration plot. |
| Affinity data for this assay | |
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