di-SFA

Procedure (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).
2. Working solution preparation
2.1 Diluent: For lipid vesicle measurements, prepare di-SFA in 20 mM HEPES buffer, pH 7.0 (22 °C), containing 100 mM K2SO4 (high K⁺ buffer) or 100 mM Na2SO4 and 1 mM K2SO4 (low K⁺ buffer), and containing 2 mg/mL β-cyclodextrin.
2.1.1 For CEM cell suspension measurements, prepare di-SFA in Ringer's solution containing 1.5 mM K⁺ and 2 mg/mL β-cyclodextrin.
2.1.2 For patch-clamp measurements of adherent A172 and L929 cells, prepare di-SFA in Ringer's solution containing 1.5 mM K⁺ and 2 mg/mL β-cyclodextrin.
2.2 Working concentration: 1 µM di-SFA is used for lipid vesicle measurements.
2.2.1 4 µM di-SFA is used for CEM cell suspension loading.
2.2.2 5 µM di-SFA is used for A172 and L929 cell loading.

3. Staining procedure
3.1 Sample types^[1]:
3.1.1 Lipid vesicles: Prepare unilamellar lipid vesicles from 10 mg egg yolk phosphatidylcholine in 0.5 mL high K⁺ buffer using an extruder, with 7 passes through a 0.2 µm filter membrane followed by 10 passes through a 0.1 µm filter membrane.
3.1.1.1 Dilute 10 µL of the resulting vesicle suspension to 1 mL in the appropriate buffer containing 1 µM di-SFA and 2 mg/mL β-cyclodextrin.
3.1.2 CEM cell suspension: Culture CEM cells in synthetic medium X-Vivo 15 at 37 °C in a humidified 5% CO2 atmosphere.
3.1.2.1 Incubate the cells in Ringer's solution containing 1.5 mM K⁺, 2 mg/mL β-cyclodextrin, and 4 µM di-SFA for 10 min.
3.1.2.2 After centrifugation at 1500 rpm for 5 min, resuspend the cells at 2 × 10⁶ cells/mL in Ringer's solution containing 1.5 mM K⁺ .
3.1.3 A172 and L929 adherent cells: Seed the cells in culture dishes with fibronectin-coated glass bottoms.
3.1.3.1 To provide antioxidant protection, dissolve astaxanthin at 2 mM in THF, add 100 µL of this solution to 20 mL DMEM under vortexing, replace the normal extracellular medium with astaxanthin-containing medium at room temperature, and incubate the cells at 37 °C for 24 h before measurement.
3.1.3.2 For dye loading, replace the medium with Ringer's solution containing 1.5 mM K⁺, 2 mg/mL β-cyclodextrin, and 5 µM di-SFA, and incubate at room temperature for 15 min.
3.1.3.3 Rinse the cells with Ringer's solution containing 1.5 mM K⁺ .
3.2 Incubation conditions:
3.2.1 For CEM cell suspension loading, incubate in Ringer's solution containing 4 µM di-SFA for 10 min.
3.2.2 For A172 and L929 cell loading, incubate in Ringer's solution containing 5 µM di-SFA at room temperature for 15 min.
3.2.3 For lipid vesicle measurements, dilute the vesicle suspension into buffer containing 1 µM di-SFA.
3.2.4 To generate a transmembrane potential in lipid vesicles, add valinomycin in ethanol solution to a final concentration of 90 nM, with a final ethanol concentration of 0.02%.
3.2.5 To generate a transmembrane potential in CEM cell suspensions, add valinomycin in ethanol solution to a final concentration of 1.5 µM, with a final ethanol concentration of 0.02%.
3.3 Washing steps:
3.3.1 For CEM cells, centrifuge at 1500 rpm for 5 min and resuspend in Ringer's solution containing 1.5 mM K⁺ .
3.3.2 For A172 and L929 cells, rinse with Ringer's solution containing 1.5 mM K⁺ .

4. Controls
4.1 In CEM cell suspensions, adding the corresponding amount of external Na⁺ does not change the I510/I605 ratio, whereas subsequent addition of external K⁺ decreases this ratio.
4.2 When the external and internal K⁺ concentrations are equal, the ratiometric response after adding valinomycin is nearly zero, ruling out a direct effect of valinomycin on probe fluorescence.

5. Detection and analysis
5.1 Instrument type: Absorption spectra and fluorescence spectra are recorded on a spectrophotometer.
5.1.1 Time-based fluorescence ratiometric measurements from lipid vesicles or cell suspensions are acquired on a fluorescence spectrophotometer equipped with two photomultiplier tubes and monochromators.
5.1.2 Combined patch-clamp and fluorescence experiments on single adherent cells are performed on an inverted microscope equipped with a patch-clamp amplifier.
5.2 Wavelength settings: For lipid vesicle measurements, set the long-wavelength channel to 605 nm, the short-wavelength channel to 527 nm, the excitation wavelength to 450 nm, and the monochromator slit to 32 nm.
5.2.1 For cell measurements, set the long-wavelength channel to 605 nm, the short-wavelength channel to 510 nm, the excitation wavelength to 450 nm, and the monochromator slit to 32 nm.
5.2.2 For patch-clamp fluorescence, restrict the excitation light with a 440 nm (10 nm) bandpass filter, use a 455 nm dichroic mirror, use a 460 nm cutoff longpass filter, separate the emitted fluorescence with a 565 nm dichroic mirror, use a 520 nm cutoff longpass filter, and acquire the emission ratio at 1 kHz.
5.3 Result analysis: di-SFA exhibits dual emission from the N* and T* bands.
5.3.1 In lipid vesicles, hyperpolarization with Vm = -118 mV increases the relative intensity of the N* band and causes a slight red shift of both bands; the ratio of the two bands increases by approximately 9.5%, corresponding to approximately 8% per 100 mV.
5.3.2 In CEM cell suspensions, valinomycin hyperpolarizes the cells and increases the relative intensity of the N* band, whereas external K+ depolarizes the cells and decreases the relative intensity of the N* band; the ratiometric response is 15% per 100 mV.
5.3.3 In patch-clamped A172 cells, the probe exhibits rapid systematic changes in the fluorescence ratio in response to depolarizing steps, with the response completed within 1 ms.
5.3.4 After loading, A172 and L929 cells exhibit orange fluorescence at the plasma membrane, and dye internalization is detectable only 2 h after the start of the experiment.