Ionomycin (Alomone, Jerusalem, Israel) was dissolved in DMSO. For in vivo treatment, Ionomycin was intraperitoneally administered to 12-week-old alpl+/− mice and alpl−/− CKO mice at a dose of 1 mg·kg−1 per day for 28 days. The control mice were treated with only the vehicle. After Ionomycin treatment, all groups of mice were healthy.
Ionomycin
Manufactured by Alomone
Sourced in Israel
Ionomycin is a calcium ionophore that facilitates the movement of calcium ions across cell membranes. It is commonly used as a research tool in cell biology and biochemistry laboratories.
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Lab products found in correlation
Fm4 64, by Thermo Fisher Scientific (4 mentions)
Pluronic acid, by Thermo Fisher Scientific (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
ω agatoxin iva, by Alomone (2 mentions)
ω conotoxin gvia, by Alomone (2 mentions)
Progesterone, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Mibefradil, by Merck Group (1 mentions)
Nnc 55 0396, by Merck Group (1 mentions)
Ca2 ionophore a23187, by Merck Group (1 mentions)
Bsa a7906, by Merck Group (1 mentions)
Ethylene glycol bis 2 aminoethylether n n n n tetraacetic acid egta, by Merck Group (1 mentions)
Dragonfly 200, by Oxford Instruments (1 mentions)
Baclofen, by Merck Group (1 mentions)
C 300, by Alomone (1 mentions)
Sta 500, by Alomone (1 mentions)
Snx 482, by Alomone (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
14 protocols using ionomycin
1
Ionomycin Treatment in Genetic Mice
2
Calcium Signaling in Sperm Capacitation
3
HeLa Cell Calcium Imaging for Temperature Estimation
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We transiently co-transfected HeLa cells stably expressing ELP-TEMP with R-GECO, a genetically encoded Ca2+ indicator37 (link) with the same transfection method mentioned above. We observed the cells with an inverted microscope (Ti-2, Nikon) equipped with a confocal unit (Dragonfly 200, Andor Technology), and the condition was the same in the cell imaging section mentioned above. We stimulated the cells with ionomycin (I-700, Alomone Labs) to the final concentration of 4 µM in DMEM/F12 (11039-021, ThermoFisher Scientific) by a home-made perfusion system. Medium temperature was 34 °C. For data analysis, we selected ROIs in the nucleus or cytoplasm of cells and used the nucleus or cytoplasm calibration curve in Fig. 3 b for estimating temperature.
4
Calcium Channel Modulation in Neurons
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Ionomycin, ω-conotoxin GVIA, ω-agatoxin IVA, and SNX-482 were purchased from Alomone labs (catalog numbers I-700, C-300, STA-500, and RTS-500 respectively). The toxins were applied for 3 min in Tyrode’s solution at concentrations of 1 µM, 400 nM, and 500 nM for ω-conotoxin GVIA, ω-agatoxin IVA, and SNX-482, respectively (Ariel et al., 2012 (link); Ermolyuk et al., 2013 (link); Hoppa et al., 2012 (link)). Baclofen was purchased from Sigma-Aldrich (catalog number B 5399) and continuously perfused at 10 µM beginning 5 min before AP stimulation (Laviv et al., 2011 (link)).
5
Fluorescent Labeling of Cells
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FM4-64, Fluo-4 AM, pluronic acid, and laminin were obtained from Life Technologies Corporation (Invitrogen). Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs. All other chemicals were of reagent grade.
6
Stimulation and Analyses of Immune Cells
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Cells were plated at 3 × 106 cells/ml in a 24‐well plate (500 μl) for western blotting and at 1.33 × 106 cells/ml in a 96‐well plate (150 μl) for ELISA and flow cytometry. Stimuli were used at the following concentrations, if not indicated otherwise: 20 μg/ml 2′‐3′‐cGAMP (synthesized in‐house, detailed synthesis instructions available upon request) for ELISA and WB, and 40 μg/ml 2′‐3′‐cGAMP for flow cytometry; 5:1 cells:beads αCD3/CD28 (Gibco, #11132D), 1 μM ABT737 (Selleck Chemicals, #S1002), 1 μM S63845 (Selleck Chemicals, #S8383), 6,000 IU/ml IFNα2a (Miltenyi Biotec, #130‐093‐874), 20 μM zVAD (R&D Systems, #FMK001), 2 μg/ml PHA (Sigma‐Aldrich, #61764), 50 ng/ml PMA (Enzo Life Sciences, #BML‐PE160‐0005), 1 IU/ml ionomycin (Alomone Labs, #I‐700), 10 ng/ml IFNγ (PeproTech, #300‐02), 10 ng/ml TNFα (PeproTech, #300‐01A), 50 IU/ml IL2 (R&D Systems, #202‐IL‐010), TPCA‐1 (R&D Systems, #2559/10), Dasatinib (Selleck Chemicals, #S1021), Tacrolimus (Astellas PHArma, #06896463), Torin1 (InvivoGen, #inh‐tor1), and 2 μΜ Thapsigargin (Sigma‐Aldrich, #T9033‐.5MG). For HT‐DNA delivery, 200 ng of HT‐DNA (Sigma‐Aldrich, #D6898‐1G) per condition was complexed with 0.5 μl of Lipofectamine 2000 (Thermo Fisher Scientific, #11668019) per condition in Opti‐MeM (Gibco, #10149832) for 5 min at RT.
7
VZV Glycoprotein Fusion Assay
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Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich). Activity of the compounds on VZV gB/gH-gL mediated cell fusion were evaluated by stable reporter fusion assay as described previously [28 (link)]. Briefly, CHO-DSP1 or MeWo-DSP1 cells transfected with equal quantities of pCAGGS-gB, pME18S-gH[TL], and pcDNA3.1-gL plasmids using Lipofectamine 2000 were harvested at 6 hrs post-transfection, and mixed with MeWo-DSP2 cells in the presence of various concentrations of the compounds prepared in two-fold serial dilutions, ranging from 10 μM to 1.25 μM. Co-culture of cells were seeded into Nunc MicroWell 96-well Optical-Bottom Plates (ThermoFisher) and incubated for 48 hrs. The activity of Renilla luciferase was read immediately after adding substrate h-Coelenterazine (Nanolight Technology). Transfection with only vehicle plasmids pcDNA3.1 (+) and pME18S, or pcDNA3.1 (+), pME18S and pCAGGS-gB served as negative control. Cell viability was measured using CellTiter-Glo Luminescent substrate (Promega). Luminescence signal was recorded using Synergy H1 Hybrid Multi-Mode Reader (BioTek). Experiments were performed at least in triplicate.
8
Polyether Ionophore Compounds Characterization
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The chemical structures and selected properties of the polyether ionophores used in the current study can be found in Fig. S1. The compounds were obtained from the following sources: calcimycin, Alomone Labs (cat no. A-600); ionomycin, Alomone Labs (I-700); narasin, Sigma-Aldrich (N1271); salinomycin, Toku-E (S002); monensin, Toku-E (M083); maduramycin, Sigma-Aldrich (34069); and nanchangmycin, Sigma-Aldrich (SML2251). Lasalocid was prepared by isolating its sodium salt from the commercially available veterinary premix AVATEC, followed by acidic extraction with H2SO4 and crystallization from ethanol, but it can also be obtained from commercial suppliers (Sigma-Aldrich, cat no. 73996).
9
Fluorescent Dye Assay for Ion Measurements
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Chemicals were obtained from the indicated sources: 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), 3,3′dipropylthiadicarbocyanine iodide (DiSC3(5)), and 4-(6-acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy) dianiline-N,N,N′,N′-tetra acetic acid tetrakis (acetoxymethyl) ester (Fluo3-AM) from ThermoFisher Scientific; ionomycin from Alomone; and all others from Sigma-Aldrich. Fluorescent dye and test compound stock solutions were prepared in Dimethyl sulfoxide (DMSO), except for those of KCl, KOH, NH4Cl, and MnCl2, which were prepared in distilled water.
10
Intracellular Calcium Regulation Assay
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