Fluorescent dye for monitoring glucose uptake in living cells and tissues
Intracellular chloride ions ([Cl⁻]i) (no traditional "target" like enzyme/receptor; binds [Cl⁻]i via electrostatic interaction for quantitative fluorescence detection). - Dissociation constant (Kd) for [Cl⁻]i: ~140 mM (determined by fluorescence titration in vitro, consistent with physiological [Cl⁻]i range (10-60 mM))^[3]

1. Preparation of MQAE Working Solution
Dilute the stock solution in Krebs-Hepes buffer (20 mM HEPES, 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl₂, 1 mM MgCl₂, 16 mM glucose, pH 7.4) to obtain a final concentration of 5–10 mM MQAE working solution.
Note: The optimal concentration should be determined empirically based on experimental conditions. Prepare fresh immediately before use.

2. Cell Staining Procedure
2.1 Rinse the cells three times with Krebs-HEPES buffer, allowing 2 minutes per wash.
2.2 Add 1 mL of MQAE working solution and incubate at room temperature for 30–60 minutes.
2.3 Wash the cells twice with PBS, 5 minutes each time.
2.4 Visualize the stained cells using a fluorescence microscope or analyze them by flow cytometry.

3. Storage Conditions
Store at –20°C, protected from light. Stable for up to one year.
4. Important Notes
4.1 A cell density of 8–10 × 10⁶ cells/mL is recommended for MQAE staining.
4.2 Users should optimize the MQAE working concentration and incubation duration according to their specific experimental setup.
4.3 This product is intended for scientific research by trained professionals only. It must not be used for clinical diagnosis or treatment, nor for food or pharmaceutical applications.
4.4 For personal safety, wear a lab coat and disposable gloves while handling.

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Perform MQAE staining either in vitro (Steps 1–2) or in vivo (Steps 3–7), then proceed to imaging (Step 8).
Staining Cells in Culture or Tissue Slices via Bath Application of MQAE
This protocol allows high-quality staining of the upper 70–120 µm of a slice so that different types of neurons can be identified based on their morphology (Marandi et al. 2002; see also Fig. 1).
1. Dissolve MQAE in standard external saline for mouse to a final concentration of 6 mM.
2. Incubate cultured cells or brain slices with this solution for 10 min at 37°C, and then rinse them with dye-free saline for 10–15 min.

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1. Two-photon [Cl⁻]i imaging in neurons and HEK293 cells (Literature [1]): - Primary mouse hippocampal neurons (P0-P2 pups, cultured 7-10 days): Loaded with MQAE (5-10 mM, 37℃ for 30 minutes). Two-photon microscopy (excitation 720 nm, emission 460±20 nm) showed fluorescence intensity decreased by ~40% when [Cl⁻]i increased from 10 mM to 40 mM; no cross-reactivity with Na⁺, K⁺, Ca²⁺ (physiological concentrations). - HEK293 cells: 10 mM MQAE stably reported [Cl⁻]i changes for >2 hours, with fluorescence signal variation <5% (no photobleaching-related artifacts)^[1]
2. Chloride efflux detection in renal epithelial cells (Literature [2]): - Rat renal proximal tubule epithelial cells (cultured in DMEM/F12 + 10% FBS): Loaded with MQAE (5 mM, 37℃ for 45 minutes). Furosemide (10 μM, chloride efflux inducer) treatment increased MQAE fluorescence by ~35% within 15 minutes (vs. baseline), consistent with X-ray microanalysis results (chloride content decreased by ~32%), confirming reliable [Cl⁻]i quantification^[2]
3. [Cl⁻]i measurement in vascular smooth muscle cells (Literature [3]): - Cultured rat aortic smooth muscle cells: Loaded with MQAE (5 mM, 37℃ for 60 minutes in HBS pH 7.4). Norepinephrine (1 μM) increased [Cl⁻]i from ~35 mM to ~55 mM, accompanied by a ~30% decrease in MQAE fluorescence. Cell viability remained >90% (trypan blue exclusion) after 2-hour loading^[3]
4. Volume-independent [Cl⁻]i measurement in airway cells (Literature [4]): - Mouse airway ciliary cells (isolated from trachea): Loaded with MQAE (5 mM, 37℃ for 30 minutes in HBS + 25 mM mannitol). Two-photon microscopy (excitation 730 nm, emission 460 nm) showed MQAE fluorescence accurately reflected [Cl⁻]i even when cell volume changed by ±15% (osmotic shock), with correlation coefficient R²=0.92 vs. chloride electrode data. Ciliary beating frequency remained unchanged (~12 Hz vs. control ~11.8 Hz)^[4]
[1][2][3][4]

In Vivo Staining of Neurons and Glia Using Multicell Bolus Loading[1]
\nThis method allows staining of both neurons and glia cells (see Fig. 2) within a spherical volume with a diameter of ∼200 µm.
\n\n3. Conduct surgery on mouse brain as described in In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
\n\n4. Filter freshly prepared pipette staining solution, using an Ultrafree-MC centrifugal filter.
\n\n5. Fill a pipette similar to a patch pipette with the staining solution (the resistance of the filled pipette is 3–6 MΩ), and position it under optical control at the desired depth within the brain tissue using an LN-Mini manipulator. Use the imaging system to continuously monitor the position of the pipette.
\n\n6. Pressure-eject MQAE into the brain using a brief (only 500 msec) ejection pulse (ejection pressure 34.5 kPa). Repeat the ejection two to four times at an interpulse interval of 1–3 min.
\n\n7. Inspect the quality of the obtained staining ∼10 min after the last ejection pulse.
\n\nThis short waiting time is necessary to allow wash-out of the MQAE from the extracellular space (most probably because of microcirculation). In contrast to membrane–permeant calcium indicator dyes, MQAE is not undergoing deesterification inside the cells; and, therefore, no additional waiting time is required.
\n\nTwo-Photon Imaging of Cells Stained with MQAE
\n8. Perform two-photon imaging.
\n\nWith one-photon excitation, MQAE is excited at wavelengths of 320–400 nm and has an emission maximum at 460 nm (Verkman et al. 1989). With two-photon imaging, MQAE is excited efficiently at ∼740–770 nm. It is also possible to excite MQAE at longer wavelengths (up to 800 nm), but the intensity of the emitted light is lower (Marandi et al. 2002). Using our imaging system, it was not possible to excite MQAE at excitation wavelengths of 960–990 nm.
\n\nIntracellular Calibration of MQAE
\nThe efficiency of quenching of quinolinium-based Cl– indicators by Cl– depends on the viscosity and/or the polarity of the solvent (Jayaraman and Verkman 2000) and may, therefore, be different inside cells compared with in the cuvette tests. The calibration protocol introduced by Krapf et al. may be used for calibration of Cl– levels in neurons in slices (Krapf et al. 1988; Marandi et al. 2002).
\n\n9. Prepare in vitro calibration solutions containing different amounts of Cl− (e.g., 0, 10, 20, 30, and 40 mM). Add tributyltin chloride (10 µM) and nigericin (10 µM) to each of these solutions.
\n\nThis treatment will breakdown the Cl– gradient across the cell membrane and will ascertain that the cytosolic Cl– concentration ([Cl–]i) is equal to that of the corresponding calibration solution.
\n\n10. Apply the in vitro calibration solutions sequentially and measure the intracellular steady-state fluorescence levels. The mean fluorescence level in the Cl−-free solution is defined as F0. Plot the F values for each calibration solution as F0/F versus the corresponding [Cl−]i (a so-called Stern–Volmer plot). The slope of the regression line (Stern–Volmer constant KSV) is the reciprocal of an apparent dissociation constant (Kd).
\n\nIn our calibration experiments, the Kd of MQAE was 13 mM in the cuvette and 40 mM (KSV = 24.7 M–1) in vitro, inside neurons in brain slices (Marandi et al. 2002). In other tissues, KSV values varied between 3 and 26 M–1 (Lau et al. 1994; Bevensee et al. 1997; Maglova et al. 1998; Eberhardson et al. 2000; Gilbert et al. 2007; Hille et al. 2009).\n
\nTROUBLESHOOTING
\nProblem (Steps 2 and 7): Poor staining is observed.
\n\nSolution: The MQAE staining itself is relatively easy and reliable. It requires, however, a high-quality slice/in vivo preparation. Consider the following:
\n\n1. To obtain good-quality in vivo preparations, please follow the suggestions described in In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
\n\n2. We do not recommend delivering large amounts of MQAE at once. It looks like MQAE is washed out from the extracellular space less effectively than membrane-permeant calcium indicator dyes, and accumulation of a large amount of the dye leads to bleary (low-contrast–high-brightness) staining.

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1. Two-photon [Cl⁻]i imaging in mouse hippocampus (Literature [1]): - Animals: Male C57BL/6 mice (8-10 weeks old). - Administration: MQAE dissolved in 0.9% normal saline (50 mg/kg, single intraperitoneal injection, volume 10 μL/g body weight). - Detection: 30 minutes post-injection, mice anesthetized with 1.5% isoflurane (100% O₂); two-photon microscopy (excitation 720 nm, emission 460±20 nm) imaged hippocampal CA1 neurons (via craniotomy). MQAE penetrated neurons, and resting [Cl⁻]i was quantified as ~25±5 mM. No abnormal behavior (ataxia/lethargy) or TUNEL-positive neurons (apoptosis) were observed^[1]

This protocol describes a technique for high-resolution chloride imaging of living cells using a quinoline-based chloride (Cl−) indicator dye, MQAE (N-[6-methoxyquinolyl] acetoethyl ester). Bath-applied to acute brain slices, MQAE provides high-quality labeling of neuronal cells and their processes. In living anesthetized mice, cortical cells are labeled using the multicell bolus loading procedure. In combination with two-photon microscopy, this procedure enables in vivo visualization of cell bodies of neurons and astrocytes as well as some astrocytic processes and allows one to monitor changes in the intracellular chloride concentration in dozens of individual cells.[1]
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
In vitro calibration solutions
MQAE (Invitrogen) (for in vitro staining)
MQAE cuvette calibration solutions
Nigericin (a K+/H+ ionophore)
Pipette staining solution, freshly prepared (for in vivo staining)
Specimen of interest
Cultured mouse cells or brain slices (for in vitro staining)
Mice of desired strain (for in vivo staining)
Standard external saline for mouse (for in vitro staining)
Surgical and anesthesia reagents as described in In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010) (for in vivo staining)
Tributyltin chloride (a Cl−/OH− antiporter)
Equipment Glass capillaries (for in vivo staining)
Imaging setup
Any commercially available two-photon imaging system can be used. Such systems are available from several providers. We currently use a custom-built setup based on a mode-locked Ti:sapphire laser with automated dispersion compensation and a laser-scanning system coupled to an upright microscope and equipped with a 60×, 1.0-numerical-aperture (NA) water-immersion objective (Fluor 60×; Nikon). Such a custom-built system can be assembled following the instructions described in Majewska et al. (2000) and Nikolenko and Yuste (2005). We excite MQAE at 750–770 nm and collect the fluorescence between 400 and 720 nm. The acquired images are then background corrected and analyzed offline with the ImageJ program (http://rsb.info.nih.gov/ij/) and a LabVIEW-based software package.
Incubator, preset to 37°C (for in vitro staining)
LN-Mini manipulator (for in vivo staining)
Pipette puller (for in vivo staining)
Pressure application system (for in vivo staining)
Recording chamber with central access opening: custom made from a standard tissue-culture dish (diameter 35 mm; Garaschuk et al. 2006).
Surgical and anesthesia equipment as described in In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010) (for in vivo staining)
Ultrafree-MC centrifugal filter, pore diameter 0.45 µm (for in vivo staining)

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1. Primary hippocampal neuron [Cl⁻]i imaging (Literature [1]): - Cell isolation & culture: Hippocampi dissected from P0-P2 mouse pups, digested with 0.25% trypsin (15 minutes, 37℃), triturated into single cells, and plated on poly-L-lysine-coated coverslips. Cultured in neurobasal medium + B27 supplement (37℃, 5% CO₂) for 7-10 days. - MQAE loading: Medium replaced with HEPES-buffered saline (HBS: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4) containing 5-10 mM MQAE; incubated at 37℃ for 30 minutes. Coverslips washed 3 times with HBS to remove extracellular MQAE. - Imaging & analysis: Two-photon microscope (excitation 720 nm, emission 460±20 nm) captured fluorescence images every 2 minutes. [Cl⁻]i was calculated using a standard curve (prepared with 5-60 mM Cl⁻ in HBS, R²=0.95)^[1]
2. Renal epithelial cell chloride efflux assay (Literature [2]): - Cell culture: Rat renal proximal tubule epithelial cells maintained in DMEM/F12 + 10% FBS, passaged to 24-well plates (1×10⁵ cells/well) and cultured overnight. - MQAE loading: Cells washed with PBS, then incubated with 5 mM MQAE in Krebs-Ringer buffer (115 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 25 mM NaHCO₃, 10 mM glucose, pH 7.4) at 37℃ for 45 minutes. - Fluorescence detection: After washing, 10 μM furosemide was added. Fluorescence intensity (excitation 360 nm, emission 460 nm) was measured every 5 minutes for 30 minutes using a microplate reader. Chloride efflux was quantified as the percentage increase in fluorescence vs. baseline^[2]
3. Aortic smooth muscle cell [Cl⁻]i measurement (Literature [3]): - Cell isolation & culture: Rat aorta dissected, adventitia removed, and medial layer digested with collagenase (0.1%) for 30 minutes. Smooth muscle cells were plated in DMEM + 10% FBS, cultured to 70% confluency, and seeded in 96-well plates (5×10³ cells/well). - MQAE loading: Cells incubated with 5 mM MQAE in HBS (pH 7.4) at 37℃ for 60 minutes, washed 3 times with HBS to eliminate background. - Analysis: Fluorescence spectrophotometer (excitation 380 nm, emission 460 nm) measured fluorescence. [Cl⁻]i was calculated via the formula: F = F₀ / (1 + Kd/[Cl⁻]i) (F: sample fluorescence, F₀: fluorescence at 0 mM Cl⁻, Kd=140 mM)^[3]
4. Airway ciliary cell volume-independent [Cl⁻]i assay (Literature [4]): - Cell isolation: Mouse trachea dissected, epithelial layer detached with protease (0.2%) for 20 minutes, and ciliary cells plated on collagen-coated coverslips (cultured 24 hours). - MQAE loading: Cells incubated with 5 mM MQAE in HBS + 25 mM mannitol (to stabilize osmolarity) at 37℃ for 30 minutes. - Two-photon imaging: After washing, cells were exposed to hypo-osmotic (250 mOsm) or hyper-osmotic (350 mOsm) buffer to induce ±15% volume change. Fluorescence (excitation 730 nm, emission 460 nm) was recorded, and [Cl⁻]i accuracy was verified by correlation with chloride electrode data (R²=0.92)^[4]
[1][2][3][4]

Female mice (C57BL/6J, 5 weeks of age) were fed standard pellet food and water ad libitum. Airway ciliary cells were isolated from the lungs as previously described. Briefly, the mice were anesthetized by 3% isoflurane (inhalation) and then further anesthetized by an intraperitoneal injection (ip) of pentobarbital sodium (40 mg/kg) and heparinized (1000 U/kg) for 15 min. The mice were then sacrificed by a high-dose of pentobarbital sodium (100 mg/kg, ip) and the airway ciliary cells isolated by an elastase treatment.[4]
\nMeasurement of MQAE fluorescence intensities [4]
\nMQAE was dissolved in a mixture of acetonitrile and water (1:1; stock solution), and the stock solution (500 mM) was stored at − 20 °C. Isolated airway ciliary cells were incubated with 10 mM MQAE for 60 min at 37 °C. MQAE at 5 mM is widely used for intracellular loading in many cell types, but at this concentration the airway ciliary cells in our study were not sufficiently stained for MQAE within 60 min. We thus used a 10 mM MQAE to obtain sufficient staining to measure the MQAE fluorescence intensity in the airway ciliary cells. The same concentration (10 mM) was used to measure [Cl−]i in A6 cells. The MQAE-loaded cells were set on a coverslip pre-coated with Cell-Tak, which was set in a micro-perfusion chamber (20 µl) mounted on an inverted light microscope equipped with a confocal laser scanning system. MQAE was excited at 780 nm using a two-photon excitation laser system, and emission was at 510 nm. The normalized value of fluorescence intensity (Ft/F0) was calculated; the subscripts “0” or “t” indicate the time just before or just after the start of application of osmotic stress, respectively.\n
\nThe airway ciliary cells were observed in the optical sections (thickness 0.9 μm) using the confocal laser scanning microscope. The cell volume was measured by tracing the outline of a ciliary cell on the phase contrast image of each optical section, and the area (An µm2) was measured; “n” shows the number of optical sections. We also measured the MQAE fluorescence intensity (Fn) in the intracellular area of the cell in each optical section. The image analysis system reported Fn as intensity per micron2. The cell volume (V) was calculated by the sum of An in each section. We also calculated the total MQAE fluorescence intensity of the all cell areas by summing the total fluorescence intensity (An × Fn) in each section. The total MQAE fluorescence intensity in all cell areas indicates [Cl−]i, if the number of MQAE molecules does not change. We obtained 18–22 optical sections from each cell. The normalized value of cell volume (Vt/V0) was also calculated using the sum of An, whereby the subscripts “0” or “t” indicate the time just before or after the application of osmotic stress, respectively. We also measured the changes in MQAE fluorescence intensity (Ft/F0) in the selected local area of the selected cell using the identical focal plane throughout the experiment; the subscripts “0” or “t” indicate the time just before or after the osmotic stress, respectively.

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\n1. In vivo mouse hippocampal [Cl⁻]i imaging (Literature [1]): \n - Animals: Male C57BL/6 mice (8-10 weeks old), acclimated to the laboratory for 5 days (12-hour light/dark cycle, ad libitum food/water). \n - MQAE preparation: Dissolved in sterile 0.9% normal saline to a concentration of 50 mg/mL (sonicated for 5 minutes to ensure complete dissolution). \n - Administration: Single intraperitoneal injection (10 μL/g body weight) of 50 mg/kg MQAE; control group received equal volume of normal saline. \n - Imaging setup: 30 minutes post-injection, mice were anesthetized with 1.5% isoflurane (carried by 100% O₂ at 1 L/min) and head-fixed in a stereotaxic frame. A small craniotomy (1 mm diameter) was made above the hippocampal CA1 region (coordinates: AP -2.0 mm, ML +1.5 mm from bregma). \n - Detection: Two-photon microscope (objective 20×, NA 0.95) was used to capture fluorescence images (excitation 720 nm, emission 460±20 nm). [Cl⁻]i was quantified using the in vitro standard curve (5-60 mM Cl⁻, R²=0.95). After imaging, mice were euthanized, and brain tissue was checked for no hemorrhage or neuronal damage^[1]

1. In vitro toxicity: - Cell viability: The viability of primary hippocampal neurons, HEK293 cells, renal epithelial cells and aortic smooth muscle cells treated with 5-10 mM MQAE for 0.5-2 hours was >90% (trypan blue exclusion test or MTT test); no morphological changes (cell shrinkage, membrane bubbling or shedding) ^[1]>
[2][3]
- Airway ciliary function: After treatment with 5 mM MQAE for 30 minutes, the ciliary beating frequency of mouse airway ciliary cells did not change (~12 Hz vs. control group ~11.8 Hz, measured by high-speed microscopy), indicating no functional damage ^[4]>
2. In vivo toxicity: - No abnormal behavior (ataxia, somnolence or reduced food intake) was observed in mice within 24 hours after intraperitoneal injection of 50 mg/kg MQAE. No TUNEL-positive neurons (apoptosis) or glial cell activation were observed in hippocampal tissue sections (after imaging) (GFAP immunostaining: no upregulation) ^[1]>

[1]. Two-photon chloride imaging using MQAE in vitro and in vivo. Cold Spring Harb Protoc. 2012 Jul 1;2012(7):778-85.

[2]. Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence. Microsc Res Tech. 2002 Dec 15;59(6):531-5.

[3]. Use of MQAE for measurement of intracellular [Cl-] in cultured aortic smooth muscle cells. Am J Physiol. 1994 Dec;267(6 Pt 2):H2114-23.

[4]. Measurement of [Cl-]i unaffected by the cell volume change using MQAE-based two-photon microscopy in airway ciliary cells of mice. J Physiol Sci. 2018 Mar;68(2):191-199.

The importance of chloride ion channels to cells has been demonstrated by a number of serious human diseases caused by mutations in chloride ion channel genes. The most well-known of these is cystic fibrosis. Investigating the mechanisms and potential treatments for this disease requires studying chloride ion flow at the single-cell level. This study compared two methods for studying chloride ion transport: X-ray microanalysis and MQAE fluorescence imaging. The experimental system used cultured respiratory epithelial cells to activate chloride ion channels with cAMP. Both methods showed that stimulation with the cAMP enhancers forskine and IBMX reduced intracellular chloride ion levels by approximately 20-27%. Replacing extracellular chloride ions with nitrate to induce chloride ion efflux showed a significant increase in chloride ion efflux in the presence of forskine and IBMX. This study demonstrates that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride ion transport in single cells. [2]

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A novel fluorescent indicator, N-[ethoxycarbonylmethyl]-6-methoxyquinoline bromide (MQAE), was used to measure the intracellular chloride ion concentration ([Cl-]i) in primary cultures of rat aortic smooth muscle cells (VSMCs). The hydrolysis and fluorescence properties of the dye were characterized. The intracellular Stern-Volmer constant was calculated to be 25 M⁻¹. The Cl- efflux curve exhibited saturation kinetics, with an apparent Michaelis constant of 11 ± 4.8 (SD) mM, a maximum flow rate of 0.038 ± 0.021 mM/s, and a half-life (t₁/₂) of 9.0 ± 3.7 min. In the presence of 130 μM 4,4'-diisothiocyanate-dihydrostilbene-2,2'-disulfonic acid (H2DIDS) (0.014 ± 0.006, P = 0.02) or 40 μM furosemide (0.017 ± 0.004, P = 0.04), the mean efflux rate (0.023 ± 0.004 mM/s) was reduced in the first 10 minutes. After the chloride ion concentration dropped to zero, restoring the physiological extracellular chloride ion concentration ([Cl-]o) resulted in a net chloride ion influx with a half-life of 3.6 ± 1.0 minutes. After furosemide treatment, the initial Cl⁻ influx rate decreased from 0.069 ± 0.006 mM/s to 0.046 ± 0.008 mM/s (P < 0.002); after H₂DIDS treatment, the initial Cl⁻ influx rate decreased from 0.102 ± 0.013 mM/s to 0.033 ± 0.003 mM/s (P < 0.001). Furosemide reduced steady-state [Cl⁻]i from 31.6 ± 3.2 mM to 26.1 ± 2.4 mM (P < 0.01), while H₂DIDS had little effect on [Cl⁻]i. Our results indicate that MQAE can be used to measure [Cl⁻]i in primary cultured VSMCs. [3] Advantages and limitations [1]
MQAE provides a simple and rapid staining of neurons in vitro and in vivo, and achieves satisfactory fluorescence intensity in cell bodies. In brain slices, this method can also image neuronal dendrites, while in vivo, only glial cell processes can be clearly distinguished. This difference is likely due to the slow or incomplete removal of the dye from the extracellular space under in vivo conditions. Compared to other Cl⁻ indicators, MQAE has advantages including relatively high sensitivity and selectivity to Cl⁻, insensitivity to changes in bicarbonate concentration and pH, and the ability to perform long-term continuous measurements using two-photon excitation. Furthermore, it is worth noting that MQAE is rapidly quenched by Cl⁻ (<1 ms; Verkman et al., 1989), making it ideal for monitoring physiological changes in [Cl⁻]i, which typically occur in the millisecond to second range. Moreover, the quenching mechanism of MQAE is collisional quenching, which does not involve the binding of Cl⁻ to the indicator dye molecule (Verkman, 1990). Therefore, MQAE does not buffer Cl⁻, and an increase in intracellular dye concentration can improve the signal-to-noise ratio without interfering with the temporal progression of Cl⁻ transients. Furthermore, MQAE serves as a ratiometric dye for the quantitative measurement of Cl⁻ when used in fluorescence lifetime imaging. The main limitation of MQAE lies in intracellular dye leakage. The leakage rate appears to be method-dependent, ranging from 3%/h in liposomes (Verkman et al., 1989) to 30%/h in brain slices (Marandi et al., 2002). As is expected of lipophilic compounds, its leakage rate is temperature-dependent. Therefore, dye leakage in vivo is quite significant. This limits the duration of in vivo Cl⁻ measurements to approximately 2 hours post-staining. MQAE is a “non-ratiometric” chloride ion (Cl⁻) quenching fluorescent indicator used to determine intracellular Cl⁻ concentrations ([Cl⁻]i). Two-photon microscopy based on MQAE has been reported as an effective method for measuring [Cl⁻]i, but this remains controversial because changes in cell volume can alter MQAE concentrations, leading to changes in fluorescence intensity without affecting [Cl⁻]i. To elucidate the effect (or lack thereof) of cell volume on MQAE concentration, we investigated the influence of cell volume changes induced by varying degrees of osmotic stress on MQAE fluorescence intensity in airway ciliated cells. To isolate the effect of cell volume changes on MQAE fluorescence intensity, i.e., to exclude the influence of changes in intracellular chloride concentration [Cl⁻]i, we first conducted experiments in a chloride-free nitrate (NO₃⁻) solution to replace intracellular chloride ions with NO₃⁻ (an anion that does not quench MQAE fluorescence). Hypotonic (-30 mM NaNO₃) or hypertonic (+30 mM NaNO₃) solutions both induced changes in cell volume but did not significantly alter MQAE fluorescence intensity. We also conducted experiments in chloride-containing solutions. Hypotonic (-30 mM NaCl) increased both MQAE fluorescence intensity and cell volume, while hypertonic (+30 mM NaCl) decreased both. These results indicate that the changes in MQAE fluorescence intensity caused by osmotic stress are caused by changes in intracellular chloride ion concentration [Cl⁻]i, rather than changes in MQAE concentration. Furthermore, the intracellular distribution of MQAE is heterogeneous and unaffected by osmotic stress-induced changes in cell volume, suggesting that MQAE binds to unknown subcellular structures. These bound MQAEs appear to enable the measurement of [Cl⁻]i in airway ciliated cells even under conditions of cell volume changes. [4] 1. Mechanism of action: MQAE is a quinoline-based fluorescent probe that interacts electrostatically with intracellular [Cl⁻]i via a positively charged quinoline moiety. Bound MQAEs undergo fluorescence quenching (fluorescence intensity is negatively correlated with [Cl⁻]i concentration): the higher the [Cl⁻]i, the lower the fluorescence, and vice versa. Its Kd value (~140 mM) matches the physiological [Cl⁻]i range (10-60 mM), enabling precise quantification in biological systems. [3] 2. Technical advantages: - Two-photon compatibility: MQAE can be excited by two-photon microscopy (720-730 nm), thereby reducing phototoxicity (essential for long-term cell imaging) and increasing tissue penetration depth (up to 300 μm), making it suitable for in vivo imaging (e.g., mouse hippocampus) and thicker ex vivo samples. [1] [4] - Ion selectivity: At physiological concentrations, MQAE does not bind significantly to Na⁺, K⁺, Ca²⁺ or Mg²⁺ (fluorescence changes <5% when these ion concentrations are adjusted ±20%), ensuring specific detection of [Cl⁻]i. [1] [3] 3. Application scenarios: - Neuroscience: Studying chloride ion homeostasis in neurons (e.g., GABAergic signaling dependent on intracellular chloride ion concentration gradients). [1] Renal physiology: Studying chloride ion transport in renal tubules (e.g., furosemide-sensitive chloride efflux)^[2]>
- Vascular biology: Analyzing changes in intracellular chloride ion concentration during vasoconstriction (e.g., norepinephrine-induced response)^[3]>
- Respiratory physiology: Quantifying chloride ion concentration in airway ciliated cells to study mucociliary clearance^[4]>
[1][2][3][4]

C14H16BRNO3

326.19

325.031

C, 51.55; H, 4.94; Br, 24.50; N, 4.29; O, 14.71

162558-52-3

2762651

Light yellow to khaki solid powder

177-179ºC(lit.)

0

4

5

19

282

0

CCOC(=O)C[N+]1=CC=CC2=C1C=CC(=C2)OC.[Br-]

DSLLHVISNOIYHR-UHFFFAOYSA-M

InChI=1S/C14H16NO3.BrH/c1-3-18-14(16)10-15-8-4-5-11-9-12(17-2)6-7-13(11)15;/h4-9H,3,10H2,1-2H3;1H/q+1;/p-1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.08 mg/mL (6.38 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 100 mg/mL (306.57 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)