Human endometrial stromal cells (HESCs) were isolated from endometrial tissues as described previously [76 (link)]. Purified HESCs were expanded in maintenance medium of DMEM/F-12 (Invitrogen, Schwerte, Germany) containing 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Invitrogen, UK) and 1% antibiotic-antimycotic solution (Invitrogen). Confluent monolayers were decidualized in DMEM/F-12 containing 2% DCC-FBS with 0.5 mM 8-bromo-cAMP (8-Br-cAMP; Sigma, Munich, Germany) with or without 10−6 M medroxyprogesterone acetate (MPA; Sigma) to induce a differentiated phenotype. Where indicated, the cells were treated with recombinant LEFTY2 (25 ng/ml; R&D Systems, Germany) as described previously [77 (link)]. Ionomycin was used at 1 μM (Sigma) and the Orai inhibitors: 2-APB, YM-58483, and MRS-1845(TOCRIS, Germany). Ishikawa cells, an endometrial epithelial-like cell line (ECACC 99040201) [28 (link), 29 (link)], were maintained in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (Invitrogen). All cells were incubated at 37 °C in a humid atmosphere maintained at 5% (vol/vol) CO2, and routinely tested for mycoplasma infection.
Ym 58483
Manufactured by Bio-Techne
Sourced in Germany, United States, United Kingdom
YM-58483 is a small molecule inhibitor that functions as an inhibitor of the calcium release-activated calcium (CRAC) channel. It is commonly used in cell biology research applications to study calcium signaling and ion channel function.
Automatically generated - may contain errors
Lab products found in correlation
Mrs 1845, by Bio-Techne (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
8 bromo camp 8br camp, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Dcc fbs, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Dcc fbs, by Merck Group (1 mentions)
Medroxyprogesterone acetate mpa, by Merck Group (1 mentions)
2 apb, by Bio-Techne (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Ml 9 hydrochloride, by Bio-Techne (1 mentions)
Menthol, by Merck Group (1 mentions)
2 apb, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
8 protocols using ym 58483
1
Isolation and Decidualization of Human Endometrial Stromal Cells
2
Pharmacological Modulators of Ion Channels
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Thapsigargin, gadolinium chloride (GdCl3), 2-APB, capsaicin, menthol, and AITC were purchased from Sigma (St. Louis, MO, United States). Synta66 was purchased from Glixx Laboratories (Southborough, MA, United States). YM-58483 and ML-9 hydrochloride were purchased from Tocris (Minneapolis, MN, United States). TG, 2-APB, synta66, YM-58483, menthol, and AITC were dissolved in DMSO; capsaicin was dissolved in ethanol as stock solutions. All of them were further diluted in the bath solution as working solutions with a final 0.1% DMSO or ethanol.
3
Modulating Inflammatory Signaling in Ventricular Myocytes
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Isoproterenol (Sigma) and TNFα (R&D) were respectively used at 100 nM and 50 ng/mL, as previously reported26 (link). Neutralizing anti-TNFR1 or anti-TNFR2 antibodies (R&D) were preincubated 1 hour at 37 °C at a final concentration of 2.5 µg/mL. All pharmacological inhibitors were preincubated for 10 min. Orai pharmacological inhibitors YM58483 (Tocris) and Synta66 (Servier) were added at 1 µM. PLA2 activating peptide (R&D) and the cPLA2 inhibitor, methyl arachidonylfluorophosphonate (MAFP) (Sigma), were respectively used at 20 µg/mL and 4 µg/mL. Lipoxygenase and cycloxygenase inhibitors, nordihydroguaiaretic acid (NDGA) and indomethacin (Sigma), were used at 1 µM. Montelukast (Sigma) was used at 2 µM. To explore ventricular myocyte resistance to oxidative stress, we applied 100 µM of H2O2 (Sigma). Semapimod (MedKoo) was used in vitro on CD11b/c cells at 10 µM.
4
Murine Microglial Cell Lines and Compounds
5
Pharmacological Modulators of Neuronal Signaling
6
Ionomycin-Induced Calcium Signaling in Spinal Cord
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ionomycin calcium salt was purchased from Tocris Bioscience (Minneapolis, MN, USA) and used per the manufacturer’s recommendations. ionomycin was dissolved in DSMO first to make a 2 mM stock concentration. ionomycin was then dissolved in 2 mM Ca2+ aCSF at a final concentration of 1, 3, or 5 µM and perfused for 1 hour. Similarly, ionomycin was dissolved in zero Ca2+ aCSF for a final concentration of 3 µM and perfused for 1 hour. After 1 hour, the ionomycin buffer was washed out with either zero or normal 2 mM Ca2+ aCSF and images were collected every 15 minutes for an additional hour. To inhibit store-operated Ca2+ entry (SOCE), we pretreated spinal cords for 30 minutes with YM-58483 (an established blocker of SOCE; 500 nM; Tocris Bioscience) prior to ionomycin perfusion in aCSF. Thapsigargin, an irreversible Sarco-Endoplasmic Reticulum Ca2+ ATPase inhibitor, was purchased from AdipoGen Life Sciences (San Diego, CA, USA) and used per the manufacturer’s recommendations. Thapsigargin was dissolved in DMSO to make a 5 mM stock solution and further diluted in 2 mM Ca2+ aCSF to make a final concentration of 1 µM to pretreat the spinal cord for 60 minutes to empty calcium stores.
7
Quantifying Intracellular Calcium Signaling
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YM-58483, KN-93, and PD98059 were purchased from Tocris (Minneapolis, MN). Cyclopiazonic acid (CPA), thapsigargin (TG), GdCl3, 2-aminoethyl diphenyl borate (2-APB), and LPS were purchased from Sigma (St. Louis, MO). They were dissolved in Milli-Q water or dimethyl sulfoxide (DMSO) as stock solutions and further diluted to final concentrations in 0.1 % DMSO.
8
Characterization of PAR2-mediated Signaling
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Trypsin (from porcine pancreas; 2 µg/ml), elastase (from porcine pancreas; 3U/ml), aprotinin (20 µg/ml), amiloride (10 µM) and nifedipine (10 µM) were purchased from Sigma-Aldrich, Belgium. The selective PAR2 activator, 2-furoyl-LIGRLO-NH2 (5 µM), store operated calcium entry (SOCE) inhibitor YM58483 (1 µM) and Phospholipase C (PLC) inhibitor U73122 (5 µM) with its negative control U73343 (5 µM) were obtained from Tocris, Bioscience, UK. The specific PAR2 inhibitor I-191 (100 nM) was purchased at Axon Medchem, The Netherlands. Ionomycin (2 µM) was used as a positive control at the end of each experiment. All stock solutions were prepared in Milli Q water, DMSO or EtOH according to the manufacturers’ guidelines.
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