研究目的
To develop a rational and practical method for calibrating intracellular potassium fluorescent probes (APG-2 and APG-4) by achieving ion equilibration using ionophores and addressing issues like cell volume changes.
研究成果
The combination of valinomycin, nigericin, gramicidin, and ouabain (VNOG) is most effective for achieving potassium equilibration and calibration of APG-4, but cell volume changes must be controlled with sucrose. APG-4 is a useful potassium indicator sensitive to protein concentration, while APG-2 is non-selective and may serve as a volume sensor. Average intracellular potassium concentrations were measured as 132 mM in U937 and 118 mM in Jurkat cells.
研究不足
The study is limited to specific cell types (Jurkat, U937, HeLa, MDCK) and may not generalize to all cell lines. APG-4 compartmentalization in adherent cells like HeLa and MDCK could not be prevented, potentially affecting measurements. The calibration method requires careful control of cell volume and ionophore effects, which might be complex and time-consuming. The computational model assumes specific cell parameters that may vary.
1：Experimental Design and Method Selection:
The study involved computational modeling to understand ion equilibration requirements, followed by experimental testing of various ionophores (valinomycin, nigericin, gramicidin, monensin, amphotericin B, ouabain) to achieve potassium equilibration in cells. Protocols were applied to calibrate APG-2 and APG-4 probes, with adjustments for cell volume changes using sucrose.
2：Sample Selection and Data Sources:
Human cell lines (Jurkat, U937, HeLa, MDCK) were cultured and used. Cells were stained with fluorescent dyes (APG-2, APG-4, DiBAC4(3), CellTrace Far Red) and treated with ionophores in calibration buffers with varying potassium concentrations.
3：List of Experimental Equipment and Materials:
Flow cytometer (FACSAria, BD Biosciences), confocal microscope (Fluoview X1000, Olympus), inverted microscope (IX81, Olympus), ionophores (e.g., valinomycin, nigericin), dyes (e.g., APG-2, APG-4 from Teflabs), sucrose, and various chemicals from suppliers like Cayman Chemical, Sigma-Aldrich.
4：Experimental Procedures and Operational Workflow:
Cells were stained with dyes, treated with ionophore combinations for 30 minutes at 37°C in calibration buffers, and analyzed using flow cytometry or microscopy. Cell volume was measured using the transmission-through-dye method. Data on fluorescence and ion concentrations were collected and analyzed.
5：Data Analysis Methods:
Fluorescence data were analyzed using flow cytometry and microscopy. Statistical significance was assessed using multiple ordinary least squares regression in R. Curve fitting to binding equations was done with SigmaPlot. Mean fluorescence intensities and standard errors were calculated.
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Fluoview X1000
Fluoview X1000
Olympus
Laser scanning confocal microscopy for fluorescence imaging.
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IX81
IX81
Olympus
Inverted microscope for transmission-through-dye volume measurements.
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DiBAC4(3)
DiBAC4(3)
AnaSpec
Fluorescent dye for measuring cell membrane potential.
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CellTrace Far Red
CellTrace Far Red
ThermoFisher Scientific
Fluorescent dye for covalent binding to intracellular amines to assess cytoplasmic material conservation.
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FACSAria
FACSAria
BD Biosciences
Flow cytometry for analyzing cell fluorescence and scatter parameters.
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APG-2
APG-2
Teflabs
Fluorescent potassium probe for intracellular ion measurements.
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APG-4
APG-4
Teflabs
Fluorescent potassium probe for intracellular ion measurements.
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Valinomycin
Cayman Chemical
Potassium ionophore used in calibration experiments.
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Nigericin
Cayman Chemical
K+/H+ exchanger ionophore used in calibration experiments.
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Gramicidin
Sigma-Aldrich
Nonspecific channel for monovalent cations used in calibration experiments.
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Ouabain
Acros Organics
Na+/K+ pump inhibitor used in calibration experiments.
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Amphotericin B
Sigma-Aldrich
Nonspecific channel for small solutes used in calibration experiments.
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Monensin
Cayman Chemical
Na+/H+ exchanger ionophore used in calibration experiments.
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Staurosporine
Cayman Chemical
Protein kinase inhibitor used in experiments to study ion changes.
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Sucrose
Sigma-Aldrich
Osmotic agent used to control cell volume in calibration buffers.
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Acid Blue 9
TCI America
Dye used in transmission-through-dye method for cell volume measurements.
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