We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells， we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for β-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.
We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells， we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for β-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.
