Computational protocol: Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3

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Protocol publication

[…] Cell surface-expressed FLAG-tagged hT1R3 and mT1r3 and their mutants were detected with flow cytometry. Stable cell lines were washed twice with ice-cold FACS buffer (PBS containing 2% FCS (BioWest, Nuaillé, France), 0.1% NaN3), then incubated for 15 min on ice with a polyclonal antibody to FLAG tag (1∶200), washed thrice with FACS buffer, and incubated for 15 min with phycoerythrin-labeled anti-rabbit IgG (1∶250; Cat. No. 12-4739­81, eBioscience Co., San Diego, CA) on ice. Cells were simultaneously stained with 7-amino-actinomycin D to eliminate dead cells. Samples were quantified on a FACSCanto II (BD Biosciences, San Jose, CA), and data were analyzed with BD FACSDiva software (BD Biosciences). Relative surface expression of FLAG-tagged hT1R3, mT1r3 and their mutants were calculated as follows: MFI (mean fluorescence intensity) of each sample/MFI of negative control sample. MFI was analyzed in 10,000 cells. G16-gust44 cells that did not express T1rs were used as negative controls. The analyses were performed in triplicate, and the results were compared with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test of significance using the SigmaPlot software (Systat Inc., Chicago, IL). […]

Pipeline specifications

Software tools BD FACSDiva, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens