Chronically activated human Th2 lymphocytes were prepared as previously described (30 (link)) and were labelled with the dye Fluo-3 (Molecular Probes, Eugene, OR) at a final concentration of 4 μM. Following washing, cells were plated at 300,000 cells per well and stimulated with chemokine at varying concentrations. Ca2+ mobilization was then measured on a 96-well FLIPR System (Molecular Devices, Sunnyvale, CA) as previously described (31 (link)).
Flipr system
Manufactured by Molecular Devices
The FLIPR system is a high-throughput screening platform designed for cell-based assays. It employs fluorescence-based detection to monitor changes in cellular parameters, enabling researchers to study a wide range of biological processes, such as ion channel and receptor activity, in a rapid and efficient manner.
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5 protocols using flipr system
1
Th2 Lymphocyte Calcium Mobilization Assay
2
Calcium Mobilization Assay in CHO-NMUR Cells
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CHO cells stably expressing rat NMURs were established as described in the Supplementary Information. The calcium-mobilization assay was performed by using the FLIPR system (Molecular Devices) as described previously14 (link).
3
Quantifying GABA-evoked GABAAR Activity
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We used the FLIPR system (Molecular Devices) to quantify GABA-evoked activity of human GABAARs. We chose a membrane potential dye (Molecular Devices) to measure changes in membrane potentials and stably transfected HEK293 cells that expressed α1, β2 and γ2. Since we observed an increase in GABA-evoked responses when transfected with γ2 transiently, we describe the cells as having a low level of γ-subunit expression, indicating heterogeneity of GABAAR compositions in the cell (α1β2 or α1β2γ2). To assay for direct agonists, fluorescence was subtracted pre- and post compound addition. To assay for PAMs, cells were treated with compound at 20 uM and then with 5 uM GABA.
4
Functional Analysis of Chemoreceptors
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Functional analysis of Tas2r and Fprs was essentially done as described in Bufe et al. (2002 Bufe et al. ( , 2015)) . HEK293T (DSMZ # ACC-635) cells were seeded with 20%-30% confluence on poly-D-lysine-coated (10 mg/mL in PBS) black 96-well mCLEAR-plates (Greiner Bio-One). Twenty-four h later, cells were transfected using jetPEI (Polyplus-transfection SA) according to the manufacturer's protocol for 96well transfections. For Ca 2+ imaging experiments, cells were cotransfected with equal amounts of plasmids encoding a chemoreceptor and a promiscuous G q -type G-protein a-subunit. G16-Gust44 was used for Tas2rs, and G16 for Fprs. Ca 2+ signals to agonist stimulation were recorded 24 h post-transfection for Tas2rs and 48 h post-transfection for Fprs using the Ca 2+ indicator dye Fluo-4/ AM (Molecular Probes) and a fluorescence imaging plate reader (FLIPR) system (Molecular Devices). Mock controls were transfected with an empty pCDNA5 vector instead of the chemoreceptor and the appropriate G-protein a-subunit. Lyophilised peptides were routinely dissolved in the Ca 2+ imaging assay buffer C1 (130 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl 2 , 5 mM glucose, pH 7.2) as 0.2-1 mM stock solutions and kept in small aliquots at À20 C until use.
5
Heterologous Expression of Fpr3 in HEK293T Cells
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An Fpr3 gene subcloned from genomic DNA of C57BL/6J mice into pcDNA3.1 (Invitrogen) was used in heterologous experiments21 (link),80 (link). HEK293T (DSMZ # ACC-635) cells were seeded at 20–30% confluence on poly-D-lysine-coated (10 µg/ml in PBS) black 96-well µCLEAR-Plates (Greiner Bio-One). Cells were transfected 24 h later using jetPEI™ (Polyplus-transfection SA) according to the manufacturer’s protocol for 96-well transfections. For Ca2+ imaging experiments, 0.125 µg of DNA plasmid encoding Fpr3 were cotransfected with equal amounts of a plasmid encoding Gα16, a promiscuous G protein α-subunit. Ca2+ signals to agonist stimulation were recorded 48 h post transfection using the Ca2+ indicator dye Fluo-4/AM (Molecular Probes) and a fluorescence imaging plate reader (FLIPR) system (Molecular Devices). Cotransfection of a plasmid encoding Gα16 with an empty vector used to subclone Fpr3 gave no agonist-evoked Ca2+ responses.
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