10 μM BECEF (Invitrogen, B1150) was added to cell culture media for 30 min at 37 °C in 5 % CO2 incubator. Cultures were washed twice and then fluorescent intensities of BCECF was determined with a SpectraMax i5 plate reader in a buffer comprised of 25 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM KHPO4, 1 mM MgSO4, 2 mM CaCl2, and 5 or 25 mM glucose. The cultures were then treated with a pH 7.7 buffer containing 10 μM Nigericin (Invitrogen, N1495), 25 mM HEPES, 105 mM KCl, and 1 mM MgCl for 5 min at 22 °C followed by determination of BCECF fluorescent intensities at high pH. The cultures were then treated with a pH 6.6 buffer containing 10 μM Nigericin (Invitrogen, N1495), 25 mM HEPES, 105 mM KCl, and 1 mM MgCl for 5 min at 22 °C followed by determination of BCECF fluorescent intensities at low pH. A linear relationship between pH 6.6 and 7.7 was observed and sample pH was estimated relative to pH standards for each individual culture well.
Nigericin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
Nigericin is a polyether ionophore compound produced by the bacterium Streptomyces hygroscopicus. It functions as an antibiotic and ionophore, enabling the transport of monovalent cations across cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Draq5, by Thermo Fisher Scientific (6 mentions)
Valinomycin, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Bcecf, by Thermo Fisher Scientific (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Vx 765, by Selleck Chemicals (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Bcecf am, by Thermo Fisher Scientific (3 mentions)
Pam3csk4, by InvivoGen (3 mentions)
Nigericin, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Staurosporine, by Cayman Chemical (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Tlrl 3pelps, by InvivoGen (2 mentions)
Llome, by Santa Cruz Biotechnology (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Becef, by Thermo Fisher Scientific (2 mentions)
50 protocols using nigericin
1
BCECF-Based Intracellular pH Measurement
2
Ratiometric BCECF Imaging for Intracellular pH
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The pHi of ArchT transfected
RPE cells and control RPE cells were directly calculated using 2′,7′-bis(carboxyethyl)-5(6)-Carboxyfluorescein
(BCECF, Biotium). BCECF is a ratiometric dual excitation/single-emission
pH biosensor (405 ex/525 em for pH insensitive fluorescence, 488 ex/525
em for pH-sensitive fluorescence). Cells were loaded with 1 μM
BCECF for 10 min, washed with complete media 3 × 5 min. We performed
ratiometric imaging of cells loaded with BCECF dye (10 min, 37 C,
5% CO2). For standardization, pH standard buffers (pH ∼6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) (a
protonophore) and added sequentially to cells to equilibrate pHe and
pHi as previously described.28 (link) Individual
cell pHi was back-calculated using single-cell standard curves generated
from ratiometric fluorescence in nigericin standard buffers and pHi
values were reported inFigure 1 B.
RPE cells and control RPE cells were directly calculated using 2′,7′-bis(carboxyethyl)-5(6)-Carboxyfluorescein
(BCECF, Biotium). BCECF is a ratiometric dual excitation/single-emission
pH biosensor (405 ex/525 em for pH insensitive fluorescence, 488 ex/525
em for pH-sensitive fluorescence). Cells were loaded with 1 μM
BCECF for 10 min, washed with complete media 3 × 5 min. We performed
ratiometric imaging of cells loaded with BCECF dye (10 min, 37 C,
5% CO2). For standardization, pH standard buffers (pH ∼6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) (a
protonophore) and added sequentially to cells to equilibrate pHe and
pHi as previously described.28 (link) Individual
cell pHi was back-calculated using single-cell standard curves generated
from ratiometric fluorescence in nigericin standard buffers and pHi
values were reported in
3
BCECF-based Intracellular pH Measurement
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10 µM BECEF (Invitrogen, B1150) was added to cell culture media for 30 min at 37 °C in 5 % CO 2 incubator. Cultures were washed twice and then fluorescent intensities of BCECF was determined with a SpectraMax i5 plate reader in a buffer comprised of 25 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM KHPO 4 , 1 mM MgSO 4 , 2 mM CaCl 2 , and 5 or 25 mM glucose. The cultures were then treated with a pH 7.7 buffer containing 10 µM Nigericin (Invitrogen, N1495), 25 mM HEPES, 105 mM KCl, and 1 mM MgCl for 5 min at 22 °C followed by determination of BCECF fluorescent intensities at high pH. The cultures were then treated with a pH 6.6 buffer containing 10 µM Nigericin (Invitrogen, N1495), 25 mM HEPES, 105 mM KCl, and 1 mM MgCl for 5 min at 22 °C followed by determination of BCECF fluorescent intensities at low pH. A linear relationship between pH 6.6 and 7.7 was observed and sample pH was estimated relative to pH standards for each individual culture well.
4
Inflammasome Activation and Inhibition
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NLRP3 inflammasome priming was performed by incubation with 100 ng/mL or 500 ng/mL LPS (O111:B4, MilliporeSigma, L4391) for 5 hours. Activation was induced by the following treatments, as indicated: 5 mM ATP (MilliporeSigma, A7699) for 30 minutes; 3 μM nigericin (Enzo, BML-CA421-0005) for 2 hours; 5 μM nigericin for up to 2 hours; 10 μM nigericin for 1 hour; 250 mg/mL MSU for 3 hours; 250 mg/mL cholesterol crystals for 3 hours; 250 mg/mL Alum crystals (Thermo Fisher Scientific, 771610) for 6 hours; 1 mg/mL flagellin (Enzo, ALX-522-058-C010) for 3 hours; 1 mg/mL poly (dA:dT) (InvivoGen, tlrl-patn) for 3 hours. MSU crystals and cholesterol crystals were made as described previously (77 (link), 78 (link)). BMDMs were treated with the following PLK1 inhibitors: 3 μM cyclapolin 9 (Tocris, 3316), 10nM SBE13 (Selleckchem, S7720), 50nM Ro3280 (Stratech, S7248-SEL), and 0.4 or 0.8 nM BI6727 (Selleckchem, S2235). A CytoTox 96 nonradioactive cytotoxicity assay (Promega, G1780) was used to measure lactate dehydrogenase (LDH) as an indication of cell viability following the manufacturer’s instruction.
5
Inflammasome Activation Pathway Protocol
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ODZ was provided by Professor Sang-Kyu Ye (Department of Pharmacology, Seoul National University College of Medicine, Seoul, Republic of Korea) [21 (link)]. Penicillin-streptomycin, trypsin-EDTA, Fetal bovine serum, RPMI 1640, DMEM-high glucose pyruvate, and Opti-MEM were purchased from Gibco (Grand Island, NY, USA). LPS (O111:B4), ATP and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin, nano-SiO2, imiquimod, poly(dA:dT), MSU crystal, Y-VAD-CMK, MCC950, lipofectamine-2000, and mouse IL-1β ELISA kit were purchased from InvitroGen (San Diego, CA, USA). Flagellin was purchased from Adipogen (San Diego, CA, USA). Mouse IL-1β antibody (AF-401-NA) and human IL-1β ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against mouse caspase-1 (AG-20B-0042-C100), ASC (AG-25B-0006-C100), and NLRP3 (AG-20b-0014-C100) were purchased from Adipogen (San Diego, CA, USA), and mGSDMD (ab209845) was purchased from Abcam (Cambridge, UK). Anti-FLAG antibody (F1804) and β-Actin antibody (SC-4778) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Santa Cruz biotechnology (Dallas, TX, USA), respectively.
6
Quantification of Inflammatory Cytokines
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Ultrapure LPS was obtained from InvivoGen, nigericin was obtained from Invitrogen, okadaic acid was obtained from Calbiochem, lethal factor and protective antigen were obtained from List Biological Laboratories, and Gene Juice was purchased from Merck. DRAQ5 was purchased from eBioscience. The ELISA kits for mouse IL-1β and mouse TNF were obtained from R&D Systems and were used according to the manufacturer’s instructions.
7
Macrophage Activation and Modulation
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THP-1 cells were induced to macrophage-like phenotype by the addition of 50 ng ml−1 PMA (Sigma Aldrich, St. Louis, MO, USA) for 16 h before cell experiments. The differentiated THP-1 cells and MPMs were set up into three groups: the negative control (Ctrl), LPS + NIG (LPS + NIG), and LPS + NIG + hucMSC-Ex (hucMSC-Ex). One microgram per milliliter LPS (Sigma Aldrich) was added to the LPS + NIG group and hucMSC-Ex group, followed by the addition of 1 μM Nigericin (Invitrogen) after 4 h, while 200 μg ml−1 hucMSC-derived exosome was added to the hucMSC-Ex group at the same time of LPS addition. After 12 h of co-cultivation, cells and supernatants were collected for subsequent analysis.
8
Inflammasome Activation Assay
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9
SO2 Donor Effects on Ion Transport
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Sodium sulfite and sodium bisulfite (Na2SO3/NaHSO3), 4,4′- diisothiocyanatostilbene -2,20-disulfonic acid (DIDS) and H89 were purchased from Sigma-Aldrich (St. Louis, MO, United States). Nigericin (N1495) and Fluo 4-AM (F14217) were purchased from Invitrogen (Eugene, OR, United States), and BCECF/AM was purchased from Thermo Scientific (Waltham, MA, United States). Dithiothreitol (DTT) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Bay K8644 was purchased from selleck (Houston, TX, United States). SO2 donor was freshly prepared using Na2SO3/NaHSO3 dissolved in deionized water in 3:1 mole ratio. DIDS, H89, BCECF/AM and Fluo 4-AM were dissolved in DMSO. Nigericin was dissolved in ethanol.
10
Ratiometric Fluorescence-based pH Measurement
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In Figure S1 , pHi increases were directly calculated from pHluorin/mCherry
ratios as previously described.11 (link) Briefly,
we took an initial acquisition of pHluorin/mCherry intensity ratios,
ran the stimulation protocol acquiring only in the pHluorin channel,
and then captured a poststimulation acquisition of pHluorin/mCherry
ratios. For standardization, pH standard buffers (pH ∼ 6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) and
added to cells to equilibrate pHe and pHi as previously described.28 (link) Individual cell pHi was back-calculated using
single-cell standard curves and reported inFigure S1C .
ratios as previously described.11 (link) Briefly,
we took an initial acquisition of pHluorin/mCherry intensity ratios,
ran the stimulation protocol acquiring only in the pHluorin channel,
and then captured a poststimulation acquisition of pHluorin/mCherry
ratios. For standardization, pH standard buffers (pH ∼ 6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) and
added to cells to equilibrate pHe and pHi as previously described.28 (link) Individual cell pHi was back-calculated using
single-cell standard curves and reported in
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