pHi was measured by SNARF-1 in cell lines (ThermoFisher Scientific), pHrodoTM Red in primary AML samples (ThermoFisher Scientific), or pH reporter mCherry-SEpHluorin as reported previously [8 (link), 11 ]. In brief, PBS-washed AML cells were incubated with SNARF-1 or pHrodoTM Red for 37 °C, washed and re-suspended in PBS. The fluorescence intensities (SNARF-1: ratio of 580 and 640 nm; pHrodoTM Red: 580 nm) were measured by CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA). NHE1 activity was analyzed as reported previously [44 ]. After incubation in SNARF-1, AML cells were treated with drugs for 4 hours and then acid loaded with 20 mM NH4Cl for 5 min followed by Na+ free Krebs’ modified buffer solution [140 mM tetramethylammonium chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 0.3 mM NaH2PO4, 10 mM HEPES, 5 mM glucose, pH 7.4] for 15 min. Thereafter, the cells were replenished with Na+ (140 mM). pHi recovery was measured for 10 min at 30-s intervals by CLARIOstar microplates reader (BMG Labtech, NC, USA).
Snarf 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
SNARF-1 is a pH-sensitive fluorescent dye that can be used to measure intracellular pH in biological samples. It exhibits a shift in emission spectrum in response to changes in pH, allowing for ratiometric measurement of pH levels.
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Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
β mercaptoethanol, by Merck Group (3 mentions)
Bcecf, by Thermo Fisher Scientific (2 mentions)
Infinite m1000, by Tecan (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Indo 1 am, by Thermo Fisher Scientific (2 mentions)
Negative b cell or cd4 t cell purification kit, by Miltenyi Biotec (2 mentions)
Phrodo red, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Fluostar omega plate reader, by BMG Labtech (1 mentions)
Nigericin, by Merck Group (1 mentions)
C1272, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
35 mm petri dishes, by BD (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Blebbistatin, by Cayman Chemical (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
26 protocols using snarf 1
1
Measuring Intracellular pH in AML Cells
2
Fluorescent Intracellular Parasite Labeling
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Parasites were stained with the intracellular dye SNARF-1 (Thermofisher) to render them fluorescent. Parasites were incubated in 6 μM solution of SNARF-1 in PBS for 30 min and subsequently washed twice. SNARF-1 stained fluorescent parasites were detected at an excitation wavelength of 488 nm and an emission wavelength of 610 nm.
3
Airway Surface Liquid pH Measurement
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pHASL was assayed as previously reported (77 (link)). Briefly, we used a ratiometric pH indicator SNARF-1 conjugated to 70 kDa dextran (ThermoFisher Scientific). SNARF-1 is a single excitation (514 nm), dual emission (580 nm and 640 nm) fluorescence pH indicator with optimal range near physiologic pH. To minimize modification of ASL composition, SNARF-1, dextran was delivered as a powder to the apical side and allowed to distribute into ASL for 1 h. Fluorescence ratios were obtained on a laser scanning confocal microscope (Zeiss LSM 880) and converted to pH values using calibration curves constructed from colorless standard pH solutions. The microscope chamber housing epithelia maintained a humidified environment at 37°C. To mimic physiologic conditions, 5% CO2 was added to the chamber atmosphere whenever the basolateral side was immersed in an containing buffer solution but removed when an -free buffer solution was used.
4
Intracellular pH Measurement in Cells
5
Coculture of Choroid Plexus and T Cells
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The aCSF that was used in this experiment comprised NaCl (120 mM), NaHCO3 (26 mM), KCl (2.5 mM), NaH2PO4 (1.25 mM), MgSO4 (1.3 mM), CaCl2 (2 mM), and glucose (10 mM). During preparation, a mixture of O2 (95%) and CO2 (5%) was bubbled into the aCSF for 20 min. Intact non-perfused CPs were isolated from untreated C57/BL6 mice and from mice expressing GFP under the UBC or Cx3cr1 promoter, incubated in the aCSF at 37°C with O2 (95%) and CO2 (5%), and cocultured with either non-activated or activated CD45.1+ Th1 cells stained with CFSE or SNARF-1 (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 24 h, the CPs were collected from the plate and underwent two vigorous washes to preserve T cells that either firmly adhered to the CP or had transmigrated into the stroma. The washed CPs were dissociated and immunolabeled for flow cytometry.
6
Intracellular pH Measurement in Cells
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Cells were loaded with either 5-(and-6)-Carboxy, Acetoxymethyl Ester, Acetate (SNARF-1; Thermo) or 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF; Thermo) ratiometric pH probes according to the manufacturer’s instructions. After loading with pH probe, cells were incubated in DMEM without phenol red that was either left unmanipulated or titrated to pH 6.0 with addition of either DMSO (vehicle) or dimethyl-αKG 5 mM. After 4 hours of incubation at 37° C with 5% CO2, fluorescence was measured using a plate reader (Tecan, Infinite M1000). The SNARF-1 probe was detected with a single excitation at 514 nm and a dual-emission ratio (580 nm and 640 nm). The BCECF probe was detected with a dual-excitation (490 nm and 440 nm) and a fixed emission wavelength of 535 nm. Background fluorescence (cells without probes) was subtracted before all calculations. Intracellular pH was quantified by a standard curve ranging from pH 5.5 to pH 7.5 using DMEM with nigericin 10 μM and valinomycin 10 μM to equilibrate intracellular pH with extracellular pH.
7
Measuring pH Changes in CF Bronchial Cells
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Differentiated CF bronchial epithelial cells under air-liquid interface condition were treated for 24 hours with vehicle alone (DMSO) or with ARN23765 (10 nM), 4172 (10 μM), or with the combination of ARN23765 plus 4172. At the end of the treatment, cells were incubated (37°C, 5% CO2 atmosphere) with 75 μl of a modified PBS solution with low buffer capacity on the apical side (37 ). The modified PBS solution had the following composition: 145 mM NaCl, 2.7 mM KCl, 0.81 mM Na2HPO4, 0.15 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, 100 μM CPT-cAMP, and 1 μM VX-770 (pH 7.35). After 3 hours, the apical fluid was recovered in a single step, and 50 μl of each sample was mixed with the pH-sensitive fluorescent probe SNARF-1 (D3304, Thermo Fisher Scientific; final concentration, 0.1 mg/ml) in a 96-well microplate. The fluorescence was measured in a FLUOstar Omega plate reader (BMG LABTECH) using single excitation (544 nm)/double emission (590 to 640 nm). The ratio of fluorescence emitted at 590 and 640 nm was then converted to pH values using a calibration curve.
8
Ratiometric Cytosolic pH Measurement
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The cytosolic pH was determined with SNARF®-1 (5-(and-6)-Carboxy SNARF™-1, acetoxymethyl ester, acetate; C1272, ThermoFisher), which is suitable for ratiometric pH measurements. SNARF®-1 was excited at 488 nm and emissions were recorded at 580 nm and 640 nm, essentially as described 73 . For calibration, U251N cells were seeded in 96-well plates, containing 10,000 cells/well. After overnight growth, samples were incubated with 5 μM SNARF®-1 in serum-free/phenol-free DMEM (45 min, 37oC). Calibration was performed for different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5) in the presence of 10 μM nigericin (Sigma) 73 . Fluorescence intensities were measured with a SPARK10M microplate reader (excitation: 485 nm, bandwidth 20 nm; emission E1: 635 nm, bandwidth 35 nm; and emission E2: 580 nm, bandwidth 20 nm). The ratio E1/E2 for fluorescence emission was plotted as a function of pH, and non-linear regression was used for curve-fitting. The cytosolic pH values of U251N cells were extrapolated from the calibration curve.
9
Automated Swine Airway Surface pH Measurement
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Individual SMG secretions were collected from swine tracheal membrane overlaid with a mineral oil. Using pH indicator, SNARF-1 (Thermo Fisher Scientific, Waltham, MA, USA) [15 (link)] and Flexstation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA), the ASL pH was evaluated automatically.
10
Fluorescent Dextran-Based pH Measurement
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pHASL was measured using a fluorescent ratiometric pH indicator, SNARF-1, conjugated to 70 kD dextran (Thermo Fisher Scientific). Additional details are reported in the supplement.
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