Nigericin, by InvivoGen - Product details

1

Cytokine Quantification from Monocytes

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For cytokine quantification, primary monocytes were seeded in 96-well plates at 5 × 10³ cells/well, in RPMI 1640, GlutaMAX medium (Thermofisher) supplemented with 10% fetal calf serum (Lonza) and incubated for 3 h in the presence of LPS (10 ng/mL, Invivogen). Primary monocytes were then treated for 1 h 30 with nigericin (5 μM, Invivogen); UCN-01 (12.5 μM, Sigma), TcdB (125 ng/mL, Abcam) or steroid catabolites at the indicated concentrations. When indicated, monocytes were treated with colchicine (1 μM, Sigma), nocodazole (5 μM, Sigma), VX-765 (25 μM, Invivogen), MCC950 (10 μM, Adipogen AG-CR1–3615) or Calyculin A (Sigma, 208851) 30 min before addition of steroid catabolites, UCN-01, TcdB or nigericin. Following the incubation, cells were centrifuged, and supernatants were collected.
To assess cytokine release, 8 × 10⁴ U937 cells per well of a 96 wells plate were exposed to 100 ng.mL⁻¹ of phorbol 12-myristate 13-acetate (PMA; InvivoGen) for 48 h and primed with LPS at 50 ng/mL for 3 h. When applicable, nigericin was used at 50 μg.mL⁻¹. Supernatant was collected at 3 h post treatment. Levels of IL-1β, IL-18 or TNF in cell supernatants were quantified by ELISA (R&D Systems). The number of replicates and independent experiments are listed in the corresponding figure legends.

Magnotti F., Chirita D., Dalmon S., Martin A., Bronnec P., Sousa J., Helynck O., Lee W., Kastner D.L., Chae J.J., McDermott M.F., Belot A., Popoff M., Sève P., Georgin-Lavialle S., Munier-Lehmann H., Tran T.A., De Langhe E., Wouters C., Jamilloux Y, & Henry T. (2022). Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism. Cell reports, 41(2), 111472.

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2

BMDM Inflammasome Activation Assay

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A macrophage-based assay was adapted from previously described protocols to assess inflammasome activation in BMDMs [60 (link)]. Frozen BMDMs were thawed and plated in 24-well or 96-well tissue culture-treated, flat-bottom plates in complete DMEM (DMEM + 2mM L-glutamine + 10% FBS + 1% Pen/Strep) at a density of 1x10⁶ or 2x10⁵ cells per well, respectively. After plating, cells were left to rest at 37°C and 5% CO₂ overnight. BMDMs were then primed using 1μg/mL Pam₃CSK₄ (InvivoGen) for 17 hours (h) or left untreated. Primed BMDMs were stimulated with DENV NS1, 5μM nigericin (Invivogen), or left untreated. After 24h, supernatants were spun down at 10,600 x g for 10 minutes, and the cell-free supernatant was collected. BMDM layers were washed twice with PBS and then lysed in RIPA buffer (150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris pH 7.4) supplemented with protease inhibitor (Roche). Supernatants and cell lysates were stored at -80°C until further analysis. For experiments involving inhibitors, the NLRP3 inhibitor MCC950 (InvivoGen) or caspase-1 inhibitor Ac-YVAD-cmk (InvivoGen) were added at indicated concentrations 30 min prior to treatment with DENV2 NS1 or nigericin.

Wong M.P., Juan E.Y., Pahmeier F., Chelluri S.S., Wang P., Castillo-Rojas B., Blanc S.F., Biering S.B., Vance R.E, & Harris E. (2024). The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection. PLOS Pathogens, 20(4), e1012167.

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3

Differentiation and Stimulation of Murine BMDMs

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Primary bone marrow-derived macrophages (BMDMs) were produced by cultivating bone marrow harvested from C57BL/6J young mice (Janvier Labs; ethics approval number DD24.1-5131/396/9, Landesdirektion Sachsen) in BMDM medium consisting of high glucose DMEM + GlutaMAX (Thermo Fisher Scientific), 10% heat-inactivated FBS (v/v; Thermo Fisher Scientific), 1% penicillin-streptomycin (v/v; Thermo Fisher Scientific) and 20% L929 conditioned media (v/v) on Corning^TM not TC-treated petri dishes (Sigma-Aldrich) for 6 days. Differentiated BMDMs were detached, seeded on hydrogels within a 24-well plate format at the concentration specified for each experimental approach and cultured for 14–18 h. For LPS priming, cells were challenged with 100 ng/ml ultrapure LPS from Escherichia coli (InvivoGen) for 4.5 h for most of the experiments and for 6 h for the gene expression experiments. For nigericin stimulation, BMDMs were treated with 10 μM nigericin (InvivoGen) for 1.5 h. For inhibitor experiments, 0.06% DMSO (v/v; Sigma-Aldrich), 10 μM blebbistatin (Sigma-Adlrich #B0560) or 10 μM Y-27632 (Sigma-Aldrich #Y0503) were used. BMDMs were pre-treated with the inhibitors for 1 h, and the inhibitors were kept in the medium during the subsequent priming with LPS priming and stimulation with nigericin.

Escolano J.C., Taubenberger A.V., Abuhattum S., Schweitzer C., Farrukh A., del Campo A., Bryant C.E, & Guck J. (2021). Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response. Frontiers in Cell and Developmental Biology, 9, 639815.

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4

Microglia Activation and Inhibition

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Microglia were plated in 6-well (Western blot, caspase-3 cell lysate ELISA; 5 × 10⁵ cells/mL), 8-well (immunofluorescence; 5 × 10⁴ cells/mL), or 96-well (DAPI, LDH assay; 2.5 × 10⁵cells/mL) plates for 24 h prior to treatment. Cells were exposed to nigericin (5.0 μM; InvivoGen cat# tlrl-nig), nigericin plus VX-765 (50.0 μM, 4 h pre-treatment; InvivoGen cat# inh-vx765-1), ATP-γ-S [100.0 μM, adenosine 5′-O-(3-thiotriphosphate), a thiophosphorylated phosphatase-resistant form of ATP; catalog no. 11162306001; Sigma-Aldrich], ATP plus VX-765 (as above), staurosporine (5.0μM; Abcam #ab120056) staurosporine plus VX-765 (as above), or vehicle control for 4 h unless otherwise indicated. Supernatants were harvested and stored at − 80 °C.

McKenzie B.A., Fernandes J.P., Doan M.A., Schmitt L.M., Branton W.G, & Power C. (2020). Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis. Journal of Neuroinflammation, 17, 253.

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5

Cytokine Quantification from Monocytes

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For cytokine quantification, primary monocytes were seeded in 96-well plates at 5 × 10³ cells/well, in RPMI 1640, GlutaMAX medium (Thermofisher) supplemented with 10% fetal calf serum (Lonza) and incubated for 3 h in the presence of LPS (10 ng/mL, Invivogen). Primary monocytes were then treated for 1 h 30 with nigericin (5 μM, Invivogen); UCN-01 (12.5 μM, Sigma), TcdB (125 ng/mL, Abcam) or steroid catabolites at the indicated concentrations. When indicated, monocytes were treated with colchicine (1 μM, Sigma), nocodazole (5 μM, Sigma), VX-765 (25 μM, Invivogen), MCC950 (10 μM, Adipogen AG-CR1–3615) or Calyculin A (Sigma, 208851) 30 min before addition of steroid catabolites, UCN-01, TcdB or nigericin. Following the incubation, cells were centrifuged, and supernatants were collected.
To assess cytokine release, 8 × 10⁴ U937 cells per well of a 96 wells plate were exposed to 100 ng.mL⁻¹ of phorbol 12-myristate 13-acetate (PMA; InvivoGen) for 48 h and primed with LPS at 50 ng/mL for 3 h. When applicable, nigericin was used at 50 μg.mL⁻¹. Supernatant was collected at 3 h post treatment. Levels of IL-1β, IL-18 or TNF in cell supernatants were quantified by ELISA (R&D Systems). The number of replicates and independent experiments are listed in the corresponding figure legends.

Magnotti F., Chirita D., Dalmon S., Martin A., Bronnec P., Sousa J., Helynck O., Lee W., Kastner D.L., Chae J.J., McDermott M.F., Belot A., Popoff M., Sève P., Georgin-Lavialle S., Munier-Lehmann H., Tran T.A., De Langhe E., Wouters C., Jamilloux Y, & Henry T. (2022). Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism. Cell reports, 41(2), 111472.

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6

Inflammatory Signaling Pathway Analysis

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Standard LPS (E. coli 0111:B4, Cat No. tlrl-eblps), ultrapure LPS (E. coli 0111:B4, Cat No. tlrl-3pelps), nigericin (Cat No. tlrl-nig), ATP (Cat No. tlrl-atpl), and MSU (Cat No. tlrl-msu) were purchased from InvivoGen (San Diego, CA, United States); the cell lysis buffer (CLB) (Cat No. 9803) was bought from Cell Signaling Technology (Danvers, MA, United States); the mouse immunoglobin IgG protein (Cat No. ab198772) was purchased from Abcam (Cambridge, CB2 0AX, United Kingdom); Protein A/G PLUS-Agarose (Cat No. sc-2003) was obtained from Santa Cruz (Santa Cruz, CA, United States); mouse IL-1β (Cat No. 88–7013), tumor necrosis factor-α (TNF-α) (Cat No. 88-7324), interleukin-6 (IL-6) (Cat No. 88-701364), and a human IL-1β (Cat No. BMS22) ELISA kit was bought from Thermo Fisher (Waltham, MA United States); and the CellTiter-Glo^® Luminescent Cell Viability Assay (Cat No. G7572) was from Promega.

Shi J., Xia Y., Wang H., Yi Z., Zhang R, & Zhang X. (2022). Piperlongumine Is an NLRP3 Inhibitor With Anti-inflammatory Activity. Frontiers in Pharmacology, 12, 818326.

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7

Inflammasome Activation Assay with iGluc

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THP-1 cells stable expressing iGluc with a Flag tag added to the C-terminal were generated and primed with PMA/LPS overnight, then stimulated with 5 mM ATP (Sigma-Aldrich), 5 μM Nigericin (InvivoGen, tlrl-nig), or indicated DRibbles for 12 h, the supernatant luciferase activity was examined with a Gaussia Luciferase Glow Assay Kit (Thermo Scientific, 16161). The cleavage iGluc was detected by western blot with anti-Flag antibody. The iGluc DNA sequence was a generous gift from Dr Veit Hornung.

Xing Y., Cao R, & Hu H.M. (2016). TLR and NLRP3 inflammasome-dependent innate immune responses to tumor-derived autophagosomes (DRibbles). Cell Death & Disease, 7(8), e2322-.

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8

Inflammasome Activation in THP-1 Cells

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THP-1 cells were seeded in 96-well flat-bottom tissue culture plates at 1 × 10⁵ cells/well in 200 µL. Cells were primed with E. coli LPS (100 ng/mL), P. gingivalis LPS (100 ng/mL), T. denticola LPS (100 ng/mL), T. forsythia LPS (100 ng/mL), P. gingivalis OMVs (100 ng protein/mL), T. denticola OMVs (100 ng protein/mL), T. forsythia OMVs (100 ng protein/mL), or left unprimed for 4 h at 37°C in a 5% CO₂ incubator. Cells were then stimulated with positive inflammasome activation controls Nigericin (10 µM) (Invivogen, USA), Silica (125 mg/mL) (US Silica, USA), or transfected with Poly(dAdT) (250 ng/mL) (Invitrogen, USA) using lipofectamine LTX (Invivogen, USA) as per the manufacturer’s instructions for a further 6 h. Primed cells were also treated with P. gingivalis, T. denticola, and T. forsythia OMVs at OMV: cell ratios of 10:1, 50:1, and 100:1 for 6 h or E. coli, P. gingivalis, T. denticola, and T. forsythia LPS (100 µg/mL) for 6 h. Cellular supernatants were collected and analyzed for IL-1β secretion by a human IL-1β ELISA Kit (Jomar Life Research, VIC, Australia) according to the manufacturer’s instructions.

Cecil J.D., O’Brien-Simpson N.M., Lenzo J.C., Holden J.A., Singleton W., Perez-Gonzalez A., Mansell A, & Reynolds E.C. (2017). Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo. Frontiers in Immunology, 8, 1017.

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9

NLRP3 Inflammasome Activation Assay

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ATP, nigericin, LPS, flagellin and poly(deoxyadenylic-deoxythymidylic) acid sodium salt (poly (dA:dT)) were purchased from InvivoGen (San Diego, USA). DMSO and MG132 was obtained from Sigma-Aldrich (Munich, Germany). MCC950 was purchased from Selleck (Houston, USA). Anti-DYKDDDDK-Tag antibody was purchased from MBL (Beijing, China). Anti-Myc-Tag, anti-HA-Tag and anti-β-actin were purchased from Proteintech (Wuhan, China). Anti-NLRP3, anti-ASC, anti-caspase-1 were obtained from AdipoGen (San Diego, USA). Anti-IL-1β and anti-NEK7 were purchased from Cell Signaling Technology (Danvers, USA). Secondary HRP-conjugated antibodies used were anti-mouse IgG, anti-rabbit IgG (Cell Signaling Technology, Danvers, USA).

Di Q., Zhao X., Lin J., Li X., Li X., Tang H., Zhang R., Xiao W, & Chen W. (2023). A new acid isolated from V. negundo L. inhibits NLRP3 inflammasome activation and protects against inflammatory diseases. Frontiers in Immunology, 14, 1174463.

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10

Innate Immune Stimuli Protocols

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Lipopolysaccharide (LPS) and poly-dAdT (pdAdT) were obtained from Sigma-Aldrich and Immunostimulatory DNA oligonucleotides were synthesized as described (8 (link)). Nigericin and ATP were from Invivogen and Sigma respectively. Polyinosinic-polycytidylic acid (poly I:C) was obtained from Invivogen. Sendai virus (Cantrell strain) was purchased from Charles River Laboratories (Wilmington, MA). Lipofectamine 2000® Transfection Reagent was from Invitrogen. GeneJuice was from Novagen (Madison, WI). Universal type I IFN and IFN-γ were from PBL Interferon Source (Piscataway, NJ) and PeproTech (315-05), respectively. S. typhimurium (SL1344 lab strain) was from M. O’Riordan. The plasmids used were p65-pCMV4, c/EBPβ-pcDNA (Addgene), pGL3-enhancer luciferase reporter (Promega). Other plasmids such as Asc in pMSCVneo (Clontech), p205-HA in pRZ-retro, Aim2-FLAG, p204-HA, p205-HA in pEF-BOS or HA-tagged ΔHIN-p205, ΔPYD-p205 and ΔH/ΔP-p205 in pMSCV-PIG (Addgene) were made in the lab.

Ghosh S., Wallareth C., Covarrubias S., Hornung V., Carpenter S.B, & Fitzgerald K.A. (2017). The PYHIN protein p205 regulates the inflammasome by controlling Asc expression. Journal of immunology (Baltimore, Md. : 1950), 199(9), 3249-3260.

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Nigericin

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123 protocols using nigericin

Cytokine Quantification from Monocytes

BMDM Inflammasome Activation Assay

Differentiation and Stimulation of Murine BMDMs

Microglia Activation and Inhibition

Cytokine Quantification from Monocytes

Inflammatory Signaling Pathway Analysis

Inflammasome Activation Assay with iGluc

Inflammasome Activation in THP-1 Cells

NLRP3 Inflammasome Activation Assay

Innate Immune Stimuli Protocols

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