Nigericin

Manufactured by Cayman Chemical

Sourced in United States

Nigericin is a laboratory reagent used for research purposes. It is a carboxylic ionophore that selectively transports potassium ions across cell membranes. Nigericin is commonly used in studies involving ion transport, cell signaling, and cellular metabolism.

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Lab products found in correlation

Rotenone, by Cayman Chemical (3 mentions) Adenosine triphosphate (atp), by Merck Group (3 mentions) Etoposide, by Enzo Life Sciences (2 mentions) Gapdh clone 14c10, by Cell Signaling Technology (2 mentions) Z vad fmk, by Enzo Life Sciences (2 mentions) Mitoq, by Cayman Chemical (2 mentions) Estrogens, by Steraloids (2 mentions) Odn tlr1 1826 1, by InvivoGen (2 mentions) Coenzyme a and acetyl coa, by Merck Group (2 mentions) Pam3csk4 tlrl pms, by InvivoGen (2 mentions) Antimycin a, by Merck Group (2 mentions) Kribb11, by Merck Group (2 mentions) Instant fly food, by Carolina Biological Supply (2 mentions) Lipopolysaccharide lps, by Merck Group (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Ca 074 me, by Merck Group (2 mentions) M csf, by Thermo Fisher Scientific (1 mentions) Mcc950, by Cayman Chemical (1 mentions)

Close spelling variants of this product

Nigericin Sodium Salt (6 citations)

27 protocols using nigericin

Bone Marrow-Derived Macrophage Inflammasome Activation

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BMDMs were prepared and cultured as described [14 (link)]. In brief, BM cells were flushed from femurs and tibias of mice and dispersed mechanically. Cell suspensions were filtered through a 200-mesh filter, and the remaining cells were collected by centrifugation at 300×g for 4 min. After centrifugation, the cells were resuspended and cultured in RIP 1640 supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF, 20 ng/ml, eBioscience) for 7 days.
For inflammasome activation, BMDMs were treated with LPS (100 ng/ml) for 4 h, followed by 30-min treatment with PGE₂ (0.1 μM) or MSC, followed by stimulation with nigericin (4 μM for 1 h, Cayman Chemical). EP4 antagonist was added 30 min prior to treatment with PGE₂ or MSC. For macrophage polarization, BMDMs were stimulated with PGE₂ or MSC for 24 h.

Wang J., Liu Y., Ding H., Shi X, & Ren H. (2021). Mesenchymal stem cell-secreted prostaglandin E2 ameliorates acute liver failure via attenuation of cell death and regulation of macrophage polarization. Stem Cell Research & Therapy, 12, 15.

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Platelet NLRP3 Inflammasome Modulation

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Blood from mice was obtained 6 hours after surgery via cardiac puncture [30 (link)]. Mice that underwent surgery in the absence of FAL were used as sham controls. Murine platelets were isolated as previously described [18 (link)]. For certain experiments, isolated platelets from WT mice were incubated for 30 minutes with an inhibitor against NLRP3 (MCC950; 100 nM, Cayman Chemical, Ann Arbor, MI) [31 (link)] or caspase-1 (YVAD; 100 nM, Calbiochem, Darmstadt, Germany) [32 (link)]. DMSO was used as a control. In other experiments, isolated platelets derived from TLR4 PF4 or TLR4 Flox control mice were treated for 30 minutes with the NLRP3 inflammasome activator Nigericin (10 μM, Cayman Chemical) [33 (link)]. Activation of caspase-1 in isolated platelets was measured using the FAM-FLICA Caspase-1 Assay Kit according to the manufacturer’s protocol (Immunochemistry Technologies, Bloomington, MN). Platelets were analyzed in a black 96-well microtiter plate using a plate reader for relative fluorescence units (RFUs).

Vogel S., Murthy P., Cui X., Lotze M.T., Zeh HJ I.I.I, & Sachdev U. (2018). TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia. Biochemical and biophysical research communications, 508(2), 614-619.

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Comprehensive Apoptosis and Inflammation Assay

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Val-boroPro (MedChemExpress, HY-13233A); AP20187 (MedChemExpress, HY-13992); Poly dA:dT (Sigma-Aldrich, P0883); Lipopolysaccharides from Escherichia (E.) coli K-235 (Sigma-Aldrich, L2018); z-VAD-fmk (R&D Systems, FMK001); recombinant mouse M-CSF (R&D Systems, 416-ML); Bacto-thioglycolate medium without dextrose (Difco, 0363-17-2); SUPERFASLIGAND Protein (Enzo Life Sciences, ALX-522-020-C005); Recombinant Murine TNF-α (PeproTech, 315-01A); nigericin (Cayman CHEMICAL, 11437); and Puromycin aminonucleoside (Focus Biomolecules, 10-2101) were purchased. YO-PRO-1 Iodide (Y3603), Blasticidin S (R21001), and Geneticin (11811023) were purchased from Thermo Fisher Scientific. CA-074 Me (4323-v), E-64-d (4321-v), and Pepstatin A (4397-v) were purchased from Peptide Institute (Osaka, Japan).

Tsuchiya K., Nakajima S., Hosojima S., Thi Nguyen D., Hattori T., Manh Le T., Hori O., Mahib M.R., Yamaguchi Y., Miura M., Kinoshita T., Kushiyama H., Sakurai M., Shiroishi T, & Suda T. (2019). Caspase-1 initiates apoptosis in the absence of gasdermin D. Nature Communications, 10, 2091.

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Caspase Activation and Pyroptosis Assays

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LPS was purchased from Santa Cruz Biotechnology, nigericin, and vildagliptin and Ac-YVAD-CMK from the Cayman Chemical Company, PMA and sitagliptin from Sigma, Ala-Pro-AFC from Bachem, saxagliptin from Toronto Research Chemicals, and Z-VAD-FMK and etoposide from Enzo Life Sciences. Val-boroPro^{45 (link)}, 1G244^{24 (link)}, FP-biotin^{15 (link)}, L-allo-Ile-isoindoline^{14 (link)}, and L-allo-Ile-thiazolidine^{14 (link)} were synthesized according to previously published protocols. For cell culture experiments, Val-boroPro was resuspended in DMSO containing 0.1% TFA to prevent compound cyclization. Antibodies used include: human caspase-1 (#2225, Cell Signaling Technology), mouse caspase-1 (clone Casper-1, Adipogen), caspase-3 (clone 8G10, Cell Signaling Technology), human caspase-4 (clone 4B9, Santa Cruz), human caspase-5 (clone D3G4W, Cell Signaling Technology), caspase-7 (clone D2Q3L, Cell Signaling Technology), human IL-1β (Clone 2805, R&D Systems), mouse IL-1β (clone D4T2D, Cell Signaling Technology), IL-1α (#AF-200, R&D Systems), IL-18 (#AF2548, R&D Systems), GAPDH (clone 14C10, Cell Signaling Technology), DPP7 (Clone 398024, R&D Systems), DPP8 (ab42076, Abcam), DPP9 (ab42080, Abcam), PARP (#9542, Cell Signaling Technology), GSDMD (NBP2-33422, Novus Biologicals), DPP4 (#11D7, GeneTex), FAP (ABT11, Millipore), and SCPEP1 (SAB2700267, Sigma).

Okondo M.C., Johnson D.C., Sridharan R., Go E.B., Chui A.J., Wang M.S., Poplawski S.E., Wu W., Liu Y., Lai J.H., Sanford D.G., Arciprete M.O., Golub T.R., Bachovchin W.W, & Bachovchin D.A. (2016). DPP8/9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis. Nature chemical biology, 13(1), 46-53.

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Synthesis and Characterization of Compound 8j

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Compound 8j was synthesized according to previously published protocols^{29 (link)}. VbP (Talabostat mesylate) was purchased from R&D Systems and was resuspended in DMSO containing 0.1% TFA to prevent cyclization. Bortezomib was purchased from LC laboratories, zVAD-FMK from Ubpbio, VX-765 from Apexbio Technology LLC, and etoposide from Enzo Life Sciences. LPS (from E. coli O111:B4) was purchased from Invivogen, Nigericin from Cayman Chemicals, Bestatin Methyl Ester from Santa Cruz, and MCC950 from AdipoGen. Antibodies used were: hCASP1 (no. 2225, Cell Signaling Technology), GAPDH (clone 14C10, Cell Signaling Technology), hGSDMD (NBP2-3342, Novus Biologicals), PARP (no. 9542, Cell Signaling Technology), hNLRP1 (AF6788, R&D Systems), hCARD8 (no. ab24186, Abcam), mGSDMD (no. ab209845, Abcam), GSDMD (no. 219800, Abcam), α-Actin (no. A4700, Sigma-Aldrich), mCD45 (no. 103108, Biolegend, FITC conjugate, clone 30-F11), mCD3 (no. 100235, Biolegend, APC conjugate, clone 17A2), rCD3 (no. 201411, Biolegend, PE conjugate, clone 1F4), rCD6 (no. 554904, BD Biosciences, FITC conjugate, clone OX-52), mCaspase-1 (AG20B-0042, Adipogen), mIL-1β (no. D4T2D, Cell Signaling Technologies), NLRC4 (no. ab201792, Abcam), and NLRP3 (no. ab210491, Abcam).

Johnson D.C., Okondo M.C., Orth E.L., Rao S.D., Huang H.C., Ball D.P, & Bachovchin D.A. (2020). DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes. Cell Death & Disease, 11(8), 628.

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Phthalate Ester Toxicology Assay

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DEHP (>99.5% pure, CAS 117-81-7), tri-octyl tri-mellitate (TOTM, > 99% pure, CAS 3319-31-1), and peanut oil (CAS 8002-03-7) were purchased from Sigma-Aldrich, (Oakville, ON). Di-octyl succinate (DOS) and di-heptyl succinate (DHPS) were synthesized as previously described [41 (link), 42 (link)].
Nigericin (>98% pure, CAS 28643-80-3) was purchased from Cayman Chemicals (Ann Arbor, MI). Phorbol 12-myristate 13-acetate (PMA, >99% pure, CAS 16561-29-8) was purchased from Abcam, (Toronto, ON). Sulforhodamine B (SRB, (CAS 222-529-8, 85% pure) was purchased from Sigma-Aldrich (Oakville, ON). Antibodies specific for the active p20kD caspase-1 protein were purchased from Adipogen (San Diego, CA) and antibodies specific for IL-1β were purchased from Novus Biologicals (Toronto, ON) or GeneTex (Irvine, CA).

Schwendt A., Chammas J.B., Maric M., Nicell J.A., Leask R, & Chalifour L.E. (2023). Exposure to the non-phthalate plasticizer di-heptyl succinate is less disruptive to C57bl/6N mouse recovery from a myocardial infarction than DEHP, TOTM or related di-octyl succinate. PLOS ONE, 18(7), e0288491.

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Corneal Epithelial Cell Culture and Treatment

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In general, pHCE cells were seeded in 12-well or 24-well plates at 5000 cells per cm² in 1 mL or 750 µL of corneal epithelial cell growth medium, respectively. Media was changed every other day until cells were 60% to 90% confluent. Cells were treated with the indicated concentration of VbP (Cayman Chemical, Ann Arbor, MI, USA), CQ31 (Rao et al. under review), bortezomib (Millipore Sigma), MG132 (Millipore Sigma), Z-VAD-FMK (Enzo Life Science, Farmingdale, NY, USA), or VX765 (Cayman) for the indicated amount of time. For NLRP3 experiments, cells were primed overnight with 1 µg/mL of LPS (InvivoGen, San Diego, CA, USA) followed by treatment with 10 µM nigericin (Cayman) for 6 hours. Adherent cells were seeded in 6-well dishes 3 days prior to priming, whereas suspension cells were seeded 1 day prior to priming. For poly(I:C) experiments, cells were transfected for 6 hours with 50 µL of Opti-MEM solution containing 1 µL Fugene HD (Promega, Madison, WI, USA) and either 10 µM VbP or 200 ng poly(I:C) HMW (InvivoGen, San Diego, CA, USA).

Griswold A.R., Huang H.C, & Bachovchin D.A. (2022). The NLRP1 Inflammasome Induces Pyroptosis in Human Corneal Epithelial Cells. Investigative Ophthalmology & Visual Science, 63(3), 2.

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Metabolic Regulation by Estrogens

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Estrogens were from Steraloids Inc. (Supplementary Table 1). KRIBB11 was from EMD Millipore (385570). Pam3CSK4 (tlrl-pms) and ODN (tlr1–1826-1) were from InvivoGen. LPS (L3024), poly IC (P0913), diethyl maleate (D97703), sodium acetate (S5636), celastrol (C0869), NAC (A7250), itaconate (I29204), oligomycin (495455), FCCP (C2920), and antimycin A (A8674) were from Sigma. MitoQ (89950), nigericin (11437), and rotenone (13995) were from Cayman. Coenzyme A and acetyl-CoA were from Sigma (C4780, A2056) and Cayman (16147, 16160). LPS, Pam3CSK4, pIC, and ODN were used at 100ng/mL, 100ng/mL, 25μg/mL, and 1μM, respectively.

Timblin G.A., Tharp K.M., Ford B., Winchester J.M., Wang J., Zhu S., Khan R.I., Louie S.K., Iavarone A.T., ten Hoeve J., Nomura D.K., Stahl A, & Saijo K. (2021). Mitohormesis reprograms macrophage metabolism to enforce tolerance. Nature metabolism, 3(5), 618-635.

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Imaging of NLRP3 Inflammasome Activation

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Cells seeded in an 8-chamber μ-slide (Ibidi, Verona, WI, USA) were primed with 1 μg/ml E. coli O111:B4 LPS (Sigma-Aldrich, St. Louis, MO, USA) for 2–4 h followed by the addition of inhibitors or other treatment as indicated, then stimulation with 1–5 mM ATP (Sigma-Aldrich). Alternately, cells were stimulated by addition of 20 μM nigericin (#11437, Cayman Chemical, Ann Arbor, MI, USA). Samples were imaged on a Nikon Ti microscope equipped with a C2si confocal scanner (Nikon Instruments, Melville, NY, USA) and Tokai Hit stage-top incubator (Tokai Hit Co., Shizuoka, Japan). Excitation laser lines were 408, 488, 561 and 639 nm and emission was collected by photomultipliers filtered for the standard DAPI, FITC, TRITC and Cy5 bandwidths. Objectives used were × 20 air 0.75 NA, × 60 oil immersion 1.4 NA or × 60 water immersion 1.2 NA, all from Nikon. Where indicated, cells were imaged in the presence of 5 μM TO-PRO-3 (Molecular Probes). For calcium imaging, cells were loaded with 1 × Fluo-4 DIRECT solution (Molecular Probes) and incubated for 30–60 min before imaging. For analysis of mitochondrial ROS production, MitoSOX red (Molecular Probes) was added 15 min after stimulation with ATP.

Yaron J.R., Gangaraju S., Rao M.Y., Kong X., Zhang L., Su F., Tian Y., Glenn H.L, & Meldrum D.R. (2015). K+ regulates Ca2+ to drive inflammasome signaling: dynamic visualization of ion flux in live cells. Cell Death & Disease, 6(10), e1954-.

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Nigericin Dose Response in Flies

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Newly hatched flies were collected and placed into vials with instant fly food (Carolina Biological Supply Company, USA) mixed with different doses (0, 2, 10 and 50 μM) of Nigericin (Cayman Chemical, 11437) from the stock solution (50 mM dissolved in DMSO). The flies were fed for 7 days and changed with fresh vial every 24 hours. After treatments, fly thoracic samples were harvested and used for sample preparation.

Li S., Wu Z., Li Y., Tantray I., De Stefani D., Mattarei A., Krishnan G., Gao F.B., Vogel H, & Lu B. (2020). Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD. Cell reports, 32(5), 107989.

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