pHi measurements were carried out as previously described (32 (link)). Briefly, cells were seeded into 96-well plates (3000 cells per well) in quadruplicates and then treated with SLC-0111, compound 13, compound 11, S0859, or NH4Cl and incubated in hypoxia for 72 hours. The pHi measurements were carried out using the Fluorometric Intracellular pH Assay Kit (Sigma-Aldrich, catalog no. MAK150) according to the manufacturer’s instructions. Briefly, growth medium was removed, and cells were loaded with 50 μl of BCFL-AM dye loading solution for 30 min at 37°C in hypoxia, followed by treatment with fresh inhibitor for 15 min. Ratiometric measurements were then carried out at λex = 505, λem = 535 and λex = 430, and λem = 535 using a SpectraMax i3x microplate reader (Molecular Devices). A calibration standard curve was prepared using pH calibration buffers (pH 6.0 to pH 8.0; increments of 0.5 pH units) containing 10 μM nigericin. Dye loading, drug treatment, and calibration were performed using Hanks’ balanced salt solution (HBSS) with 20 mM Hepes, without sodium bicarbonate, provided with the kit. A sigmoidal 4PL nonlinear regression model was used to fit the calibration curve and interpolate the experimental pHi values.
Fluorometric intracellular ph assay kit
Manufactured by Merck Group
The Fluorometric Intracellular pH Assay Kit is a laboratory instrument designed to measure the pH levels inside cells. It utilizes fluorescent dyes that change their emission spectra in response to changes in the intracellular pH, allowing for accurate monitoring of the pH within the cellular environment.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Spectramax i3x microplate reader, by Molecular Devices (1 mentions)
Infinite 200 microplate reader, by Tecan (1 mentions)
Colorimetric assay kit, by Abcam (1 mentions)
Atp bioluminescence assay kit cls 2, by Roche (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
4 protocols using fluorometric intracellular ph assay kit
1
Intracellular pH Measurement in Hypoxic Cells
2
Intracellular pH Measurement Assay
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Intracellular pH (pHi) measurements were carried out using the Fluorometric Intracellular pH Assay Kit (MAK150, Sigma) according to the manufacturer’s instructions. MDA-MB-231, MCF-7, and HFFF2 cells were seeded into 96-well plates (40,000 cells/well) in culture media. After 24 h of incubation, the growth medium was replaced with 100 µL of HBBS buffer. Then, dye loading solution was added (100 µL/well) and incubated protected from light in a 5% CO2/95% air, 37 °C incubator for 30 min. Subsequently, a solution of MEL, nanoparticles, and complexes in HBBS buffer was added (50 μL/well) to obtain final concentrations 20 μg/mL for nanoparticles and 10 μg/mL for MEL. Immediately after adding the compounds, fluorescence was measured at λex = 490/λem = 535 nm (cut off at 515 nm) using a Tecan Infinite 200 microplate reader (Tecan, Durham, NC, USA).
3
Redox Metabolism Profiling of E. coli
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Relative ATP measurement was done using ATP Bioluminescence Assay Kit CLS II, Roche. ROS levels were detected by H2DCFDA and DHR123 fluorescent dyes (Thermo-Fisher). We used DHR123 probe along with 10 µM ferrous ammonium sulfate to detect extracellular H2O2. 10 mM of each ROS quenchers (tiron, thiourea and sodium pyruvate) were used. The fluorometric intracellular pH assay kit (Sigma) was used to monitor intracellular pH change. Colorimetric assay using methylene blue has been performed in a tightly sealed microfuge tube containing growing E. coli cells in the aerated LB medium. Reazurine dye has been added in the aerobically growing bacterial culture to see the color change. Colorimetric assay kits (Abcam) were used to detect relative levels of pyruvate, NAD, NADH in the manganese-fed, (manganese + iron)-fed and unfed cells. Aconitase, catalase, NDH-1, SDH, and glutamate synthase (GS) assays were performed, as described54 (link)–57 (link). Relative ubiquinone level in the cells was determined as described by Chehade et al.58 .
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Intracellular pH Measurement Protocol
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