Calcium 5 assay kit

Intracellular calcium flux was measured using a fluorometric imaging plate-reader (FLIPR) Calcium 5 Assay Kit and FlexStation 3 Microplate Reader (Molecular Devices, San Jose, United States). Calcium 5 dye (90 μL) prepared in low potassium HBSS (Thermo Fisher Scientific) containing (in mM) 145 NaCl, 22 HEPES, 0.338 Na₂HPO₄, 4.17 NaHCO₃, 0.441 KH₂PO₄, 0.407 MgSO₄, 0.493 MgCl₂, 1.26 CaCl₂, 5.56 glucose, and 250 probenecid with pH 7.4 was applied to each well and incubated for 1 h at 37°C, ambient air prior to testing.
Fluorescence readings were taken at 2 s intervals (λ_excitation = 485 nm, λ_emission = 525 nm). Following 2 min baseline reading, 20 μL of SB2193, SB2193F, or NNC 55-0396 (10 nM—10 μM) was added to cells and read for 5 min, then CaCl₂ (10 mM) was added and read for 3 min. Baseline fluorescence averaged over the 20 s interval preceding CaCl₂ addition was subtracted and normalized to the vehicle response. Changes in fluorescence were measured in relative fluorescence units (RFU). All compounds were assayed in ≥ five independent experiments (n = 5–8) performed in duplicate. Order of compound addition and location on the plate were varied between replicates. Data was fit to a four-parameter non-linear dose-response curve using GraphPad Prism 8.2.0 software (La Jolla, United States) to determine IC₅₀ values.

CHO cells transfected with wild-type or mutated B2R and wild-type or mutated G_q were seeded into 96-well black plates at a density of 30,000 cells per well and incubated for 24 h. Then cells were loaded with reagents from Calcium-5 Assay Kit (Molecular Devices) for 45 min at 37 °C in 5% CO₂ according to the manufacturer’s protocol. Cells were treated with varying concentrations of kallidin and detected with Flexstation 3 Multi-Mode Microplate Reader (Molecular Devices) with excitation at 485 nm and emission at 525 nm. Data were analyzed by GraphPad Prism 5 and presented as Mean ± S.E.M. from three independent experiments.

Briefly, CHO‐Gα16 cells individually overexpressing EP1 (human), EP2 (human), EP3 (human), and EP4 (human, monkey, dog, rat, and mouse) were plated into 96‐well‐black plates at a density of 2 × 10⁴ cells/100 μl per well and incubated overnight. On the following day, the plate was loaded with the reagents (100 μl/well) of the Calcium‐5 Assay Kit (Molecular Devices, San Jose, CA, USA) for 45 min at 37°C according to the manufacturer’s instructions. After pretreating with varying concentrations of TP‐16 for 15 min at room temperature, cell plates were treated with PGE₂ (EC₈₀) and analyzed using the Flexstation® 3 Multi‐Mode Microplate Reader (Molecular Devices). Florescence measurement data were continuously recorded for 90 s (Ex wavelength = 485 nm; Em wavelength = 525 nm).

Calcium flux was performed as described in our previous studies. Briefly, CHO cells were co-transfected with wild-type or mutant H₃R and G_qi5 using Lipofectamine 2000 according to the manufacturer’s manual. Transfected cells were seeded into a 96-well flat clear bottom black plate with a density of 25,000 cells per well and cultured overnight. Subsequently, cells were loaded with calcium dye solution from Calcium 5 assay kit (Molecular Devices) in Hanks’ balanced salt solution (20 mM HEPES, 2.5 mM probenecid in HBSS), and incubated at 37 °C for 45 min. Various concentrations of compounds were dispensed into the wells via a Flexstation III instrument (Molecular Devices). The intracellular calcium flux was detected immediately using the Flexstation III instrument (excitation at 485 nm, emission at 525 nm). Data were representative of three independent experiments and analyzed using GraphPad Prism 9.3.1.

The CXCR2 receptor activity of the heterodimer was determined using a Ca²⁺ release assay, as described previously [41 (link)]. Ca²⁺ levels were measured using a FlexStation III microplate reader using the Calcium 5 assay kit (FLIPR, Molecular Devices). Differentiated HL60 cells expressing CXCR2 were incubated with varying concentrations of either WT CXCL1, WT CXCL7, a mixture of both WTs, or the trapped CXCL7-CXCL1 heterodimer. Changes in fluorescence of the Calcium 5 dye upon addition of chemokine were measured every 5 s for up to 500 s, and the agonist response was determined from the maximum change in fluorescence. EC₅₀ values were calculated based on the response over a range of concentrations.

Modified Eagle’s medium (MEM), StemPro-34 SFM Medium and trypsin were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Sciencell (Carlsbad, CA, USA). Recombinant human stem cell factor was purchased from Peprotech (Rocky Hill, NJ, USA). Compound 48/80 (C48/80), l-glutamine, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-nylte-trazolium bromide (MTT), p-nitrophenyl-N-acetyl-β-d-glucosaminide and poly-d-lysine hydrobromide (PDL) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Triton X-100 was obtained from Amresco (Solon, OH, USA). Penicillin and streptomycin were purchased from Beyotime Biotechnology (Shanghai, China). Ginsenoside Rd, Rg1, Rf, 20(S)-Rg3 and 20(R)-Rg3 were purchased from Must Bio-technology (Chengdu, China). Pluronic acid F-127 was obtained from Invitrogen (Carlsbad, CA, USA). A calcium 5 assay kit was obtained from molecular devices (Sunnyvale, CA, USA). ELISA kits for histamine were purchased from Biocalvin (Suzhou, China). An Annexin V-FITC fluos staining kit was purchased from KeyGEN BioTECH (Nanjing, China).

Calcium flux assay was conducted as described in our previous study [30 (link)]. Briefly, CHO-Gα16 cells were transfected, respectively, with human EP1, human EP2, human EP3, human EP4, monkey EP4, mouse EP4 and rat EP4 overexpression constructs using lipofectamine 2000 according to the manufacturer’s manual. Cells were then seeded into a 96-well black plate with a cell density of 2 × 10⁴ cells/100 μL per well for a 16-h culture. On the next day, cells were incubated for 45 min with reagents (100 μL/well) of Calcium-5 Assay Kit (Molecular Devices, San Jose, CA, USA). Then, cells were pre-treated with indicated concentrations of L001 for 15 min and stimulated with PGE₂ (EC₈₀). The calcium flux was detected subsequently by a Flexstation^® 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).