Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.
Ionomycin
Manufactured by Adipogen
Sourced in United States
Ionomycin is a calcium ionophore that facilitates the movement of calcium ions across cell membranes. It is commonly used as a laboratory tool in cell biology research to study calcium signaling and its role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Version 10, by FlowJo (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Thermo Fisher Scientific (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Cyclopiazonic acid, by Bio-Techne (1 mentions)
4 4 diisothiocyano 2 2 stilbenedisulfonic acid dids, by Merck Group (1 mentions)
Antibiotic antimycotic, by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Clodronate, by Merck Group (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
Brefeldin a, by Cayman Chemical (1 mentions)
Hek293t cells, by Takara Bio (1 mentions)
Mc3t3 e1 cells, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Fujifilm (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Mc3t3 e1 cells, by RIKEN BioResource Center (1 mentions)
7 protocols using ionomycin
1
Calcium Signaling Pathway Modulation
2
Flow Cytometric Analysis of CXCL12-Induced Calcium Flux
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Cells were harvested and resuspended in 5mL calcium loading buffer (RPMI + 25mM HEPES) at a density of 2 x 106 cells per ml. Cells were labeled with 1μM Fluo-4AM (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C in the dark with gentle agitation every 10 minutes. Following incubation, samples were washed with calcium loading buffer, centrifuged for 5 minutes at 1000 rpm, and then resuspended in 600 μl calcium loading buffer. Cells were analyzed by flow cytometry, with cells collected for 20 seconds to establish a baseline, and then samples were treated with 100 ng/ml CXCL12 and cells collected for an additional 80 seconds. The calcium ionophore ionomycin (Adipogen, San Diego, CA) was used at 1 mg/ml as a positive control for Fluo-4 loading, and PBS served as a vehicle control. Data was analyzed using FlowJo software (FlowJo, Ashland, Oregon).
3
Flow Cytometric Analysis of PBMC Responses to PRRSV
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PBMCs were plated at 500,000 cells/well and allowed to rest overnight. The following day, cells were stimulated with either media (MOCK), NC174, NADC20, NADC30, or VR2332 (MOI of 0.1); Phorbol 12-myristate 13-acetate (PMA, 5 ng/mL, Alfa Aesar, Ward Hill, MA, USA)/Ionomycin (500 ng/mL, AdipoGen, San Diego, CA, USA) was used as a positive control. Plates were cultured for 18 h; Monensin (5 μg/mL, Alfa Aesar) was added for the last 4 h of culture. Eight replicates were then pooled and stained for flow cytometry analysis according to Table 1 . Data were acquired on a Cytoflex using the CytExpert software (Beckman Coulter). Data analysis was performed with FlowJo version 10.5.3 (FLOWJO LLC) with gates based upon the FMO controls.
4
PBMC Stimulation and Flow Cytometry
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The frozen PBMCs were thawed and seeded at a density of 500,000 PBMCs per well in quadruplicate in RPMI-1640 supplemented with 10% FBS and 1X antibiotic-antimycotic (Corning). The cells were stimulated with one of four treatments: Media, MLV, LP, or HP PRRSV virus (MOI of 0.1). Phorbol 12-myristate 13-acetate (PMA, 5 ng/mL, Alfa Aesar, Ward Hill, MA, USA)/Ionomycin (500 ng/mL, AdipoGen, San Diego, CA, USA) stimulation served as the positive control. The plates were cultured for 18 h and Monensin (5 µg/mL, Alfa Aesar) was added for the last 4 h of culture. The replicates were then pooled and stained in accordance with Figure S4 . The cells were recorded on a Cytoflex using the CytExpert software (Beckman Coulter). The data analysis was performed with FlowJo version 10.5.3 (FLOWJO LLC) with gates based upon the FMO controls.
5
Osteoblast Differentiation and Clodronate Treatment
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MC3T3‐E1 cells, a mouse calvarial osteoblast cell line, were obtained from Riken Bio Resource Center (Tsukuba, Japan). MC3T3‐E1 cells were maintained in α‐minimal essential medium (12571‐063; Gibco‐BRL, Grand, Island, NY, USA) supplemented with 10% FBS (Biosera Nuaillé, France) and 100 U·mL−1 penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Osteoblast differentiation was induced by culturing cells in an osteogenic medium containing 50 μg·mL−1 ascorbic acid (FUJIFILM Wako Pure Chemical Corporation) and 10 mm β‐glycerophosphate (Sigma‐Aldrich, St. Louis, MO, USA) for 7 days. Cells were treated with 1, or 10 μm clodronate (Sigma‐Aldrich). Ionomycin (AdipoGen Life Sciences, San Diego, CA, USA) treatments were performed at 1 μm . Brefeldin A (Cayman Chemical, Ann Arbor, MI, USA) was used at 10 μm . HEK293T cells (Takara Bio, Shiga, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM) (12320‐032; Gibco‐BRL) supplemented with 10% FBS (Biosera Nuaillé) and 100 U·mL−1 penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation).
6
NLRP3 Inflammasome Activation in LPS-Stimulated Cells
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LPS from Escherichia coli O111:B4 was purchased from Sigma–Aldrich. The cathepsin B inhibitor Ca074Me was obtained from the Peptide Institute. Nigericin, ionomycin, and the NLRP3 inhibitor, MCC950, were obtained from AdipoGen Life Sciences. The caspase-1 inhibitor Z-YVAD-fmk and pan-caspase inhibitor Z-VAD-fmk were purchased from Abcam. The actin polymerization inhibitor cytochalasin D was obtained from Cayman Chemical and was used as a phagocytosis inhibitor. All inhibitors were dissolved in DMSO.
7
Investigating Protein Regulation in HEK293 Cells
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