Mn²⁺ Quenching Assay for Store-Operated Calcium Entry

Abstract

Store-operated Ca²⁺ entry (SOCE) pathway plays important roles in many cellular processes, which is largely studied by using fluorescent Ca²⁺ indicator, Fura-2. Extracellular Mn²⁺ is able to cross the plasma membrane through SOCE and quenches the fluorescence signals from Fura-2. Thus, the fluorescence quenching rate by Mn²⁺ composes a convenient assay to monitor the extent of SOCE. This chapter describes an experimental method of Mn²⁺ quenching assay for both cultured esophageal epithelial and skeletal muscle cells. It also explains how to perform a quantitative analysis of graded SOCE.

1. Introduction

Store-operated Ca²⁺ entry (SOCE) is important for many cellular processes, including proliferation, migration, gene transcription, and muscle contraction [1, 2]. In this pathway, intracellular Ca²⁺ release either through IP₃ receptor or ryanodine receptor stimulated by a variety of agonists results in reduction of Ca²⁺ concentration in the lumen of endoplasmic or sarcoplasmic reticulum (ER/SR); the depleted Ca²⁺ stores in turn activate the SOCE channel to allow extracellular Ca²⁺ across the plasma membrane, which eventually refills the empty ER/SR Ca²⁺ stores [3]. A large body of knowledge about the functional properties of SOCE has been gained by fluorescence-based intracellular Ca²⁺ measurements using Fura-2, a ratiometric Ca²⁺ indicator [4]. To gain cell membrane permeability, Fura-2 is conjugated to acetoxymethyl ester (AM), and it remains in this nonfluorescent form [5]. After entering cells, Fura-2-AM is cleaved by intracellular esterase and converted into its fluorescent form. With emission wavelength at 510 nm, Fura-2, upon Ca²⁺ binding, presents not only a dramatic increase in fluorescence intensity but also a blueshift of excitation wavelength. Thus, the ratio of fluorescence with excitation wavelengths at 340 nm for a peak of Ca²⁺-bound form and 380 nm for a Ca²⁺-unbound form is commonly recorded to directly monitor the dynamics of intracellular Ca²⁺. This ratiometric readout avoids some experimental caveats, such as uneven loading, leakage, and photobleaching of fluorescent dye, and the issues that occur when measuring Ca²⁺ in the cells with uneven thickness of cell membranes or walls.

To further isolate the activity of unidirectional SOCE from intracellular Ca²⁺ release and Ca²⁺ extrusion on the simple intracellular Ca²⁺ measurement using Fura-2, Mn²⁺ quenching assay is employed [4]. Mn²⁺ is known to be able to permeate into cells via SOCE, while it circumvents the surface membrane extrusion mechanisms and translocation into ER by Ca²⁺ pumps. Since Mn²⁺ exhibits a much higher affinity to Fura-2 than Ca²⁺, Mn²⁺ influx leads to quenching of Fura-2 fluorescent signal. As the quenching rate of fluorescence is proportional to the amounts of Mn²⁺ that enter the cells through SOCE, Mn²⁺ quenching assay represents a convenient and selective method to determine the activity of SOCE. This assay can analyze both cell populations and single cells using a cuvette-based spectrofluorometer or a microscope-based system, respectively. This assay is also suitable for high-throughput screening.

2. Materials

Prepare all solutions using ultrapure water, which reaches a resistivity of 18.2 MΩ·cm at 25 °C (e.g., prepared by Milli-Q Water Purification System, EMD Millipore, MA), and analytical grade reagents. Prepare and store all reagents at room temperature unless indicated otherwise.

2.1. Solutions

1. 10 × balanced salt solution (BSS) stock: 1400 mM NaCl, 28 mM KCl, 20 mM MgCl₂, 120 mM glucose, 100 mM HEPES, pH 7.2.

   Add about 300 mL water to a 1 L glass beaker, followed by 40.9 g NaCl, 1.05 g KCl, 2.04 g MgCl₂, 10.8 g glucose, and 11.9 g HEPES (see Note ¹). Mix well and adjust pH with NaOH to pH 7.2. Make up to 500 mL with water. Filter the solution with 0.22 μm sterile vacuum filtration system (500 mL Steritop bottle top filter unit, EMD Millipore, MA). Store at +4 °C.

2. 1 M CaCl₂ solution: Weigh 7.35 g CaCl₂ and dissolve in water up to 50 mL.

3. 0.5 M EGTA solution: Weigh 9.5 g EGTA into 30 mL water and stir with a magnetic stir bar. Add drop by drop of 1 M NaOH just enough to dissolve EGTA completely. Add water to a final volume of 50 mL (see Note ²).

4. Thapsigargin (TG): 5 mM in DMSO. Store at −20 °C protected from light.

5. Fura-2-AM: Dissolve into DMSO as 10 mM stock. Store at −20 °C protected from light (see Note ³).

6. 2-Aminoethoxydiphenylborane (2-APB): 100 mM in DMSO. Store at +4 °C under desiccating conditions.

7. 0.5 M MnCl₂ solution.

8. 20 mM N-benzyl-p-toluene sulfonamide (BTS) stock in DMSO.

2.2. Cell Culture

1. RPMI 1640/Ham’s F-12 mixed (1:1) medium containing 5% fetal bovine serum and 1% penicillin/streptomycin. Store at +4 °C.

2. DMEM complete medium containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin. Store at +4 °C.

3. DMEM differentiation medium containing 2.5% horse serum and 1% penicillin/streptomycin. Store at +4 °C.

4. Glass bottom dishes (Cat.# P35G-1.5–14-C, MatTek, MA). The central part is No. 1.5 cover glass.

2.3. Equipment

1. Ratio imaging spectrofluorometer (e.g., Photon Technology International, NJ): xenon arc lamp, computer-controlled high-speed random access monochromator, cuvette-based emission monochromator, inverted fluorescence microscope (e.g., Nikon Eclipse TE200; Nikon, Japan), S Fluor 40×/1.30 oil objective (compatible for efficient UV transmission), and CCD camera (e.g., CoolSnap ES; Roper Scientific, GA).

3. Methods

3.1. Cell Culture

1. All cells are grown in glass-bottom dishes.

2. KYSE-150, a human esophageal squamous cell carcinoma (ECSS) cell line, is cultured in 5% CO₂ atmosphere at 37 °C in RPMI 1640/Ham’s F-12 mixed medium.

3. C2C12, a mouse myogenic cell line, is cultured in 5% CO₂ atmosphere at 37 °C in DMEM complete medium.

4. After C2C12 cells reach confluent, the culture medium is changed to DMEM differentiation medium. The C2C12 myo-blasts will fuse and differentiate into myotubes after 4 days.

3.2. Solutions and Dye Loading

1. Prepare the following solutions freshly just before the experiment:

   1. BSS-EGTA: Take out 5 mL 10× BSS stock solution in tissue culture hood, and fill up to 50 mL with water. Add 5 μL 0.5 M EGTA to generate 0.5 mM final concentration of EGTA.
   2. BSS-Ca: Take out 5 mL 10× BSS stock solution in tissue culture hood, and fill up to 50 mL with water. Add 10 μL 1 M CaCl₂ to generate 2 mM final concentration of CaCl₂.
   3. BSS-EGTA-TG: Take out 5 mL BSS-EGTA solution, and add 1 μL TG to generate 10 μM final concentration of TG.
   4. BSS-Ca-2APB: Take out 10 mL BSS-Ca solution, and add 10 μL 2-APB to generate 100 μM final concentration of 2-APB.
   5. BSS: Take out 5 mL 10× BSS stock solution in tissue culture hood, and fill up to 50 mL with water.
   6. BSS-Mn: Take out 10 mL BSS solution, and add 10 μL MnCl₂ to generate 0.5 mM final concentration of MnCl₂.
   7. BSS-Mn-2APB: Add 10 μL 2-APB into 10 mL BSS-Mn to generate 100 μM final concentration of 2-APB.
   8. Triton X-100: 0.1% in BSS-EGTA.

2. Remove the medium and rinse the cells with BSS-Ca once.

3. Add 1 mL BSS-Ca solution containing 5 μM Fura-2-AM into the dish, and wrap the whole dish with foil to protect from light.

4. Incubate the cells for 40 min at 37 °C in cell culture incubator (see Note ⁴).

5. Take out the dishes and leave them for an additional 15 min period at room temperature to complete ester hydrolysis. Wash the cells with BSS-Ca twice.

3.3. Intracellular Ca²⁺ Measurement

1. Mount the dish in which KYSE-150 cells are cultured on the stage of inverted microscope, which is connected to PTI spec-trofluorometer, with excitation wavelengths at 350 and 385 nm and emission at 510 nm (see Note ⁵).

2. Set up a gravity perfusion system with four different solutions: BSS-Ca²⁺, BSS-EGTA, BSS-EGTA-TG, and BSS-Ca-2APB. Computer-controlled automated perfusion systems can also be used.

3. Position the opening tip of the perfusion system at 10× magnification until the tip is right above the region of interest at a 45° angle.

4. Choose emission wavelength as 510 nm and excitation wavelength as 350 nm and 385 nm. After drawing regions of interest (ROIs) around individual cells, simultaneously record the fluorescence intensities at 510 nm with excitation wavelengths at 350 and 385 nm. Ratio (F_{350 nm}/F_{385 nm}) is displayed in the third window (see Note ⁶).

5. Change the perfusion solutions in the specified order: BSS-Ca²⁺ (30 s), BSS-EGTA (30 s), BSS-EGTA-TG (900 s), BSS-Ca (150 s), and BSS-Ca-2APB (300 s) (Fig. 1).

6. From the traces of ratio (F_{350 nm}/F_{385 nm}), several parameters regarding intracellular Ca²⁺ homeostasis can be determined: (a) resting intracellular Ca²⁺, (b) TG-releasable ER Ca²⁺ stores, and (c) SOCE (Fig. 1).

Fig. 1.

Intracellular Ca²⁺ measurement reveals SOCE in KYSE-150 cells. The initial ratio of F350/F385 represents resting intracellular Ca²⁺ (a). Addition of TG (indicated by first arrow) in BSS-EGTA bath solution induces passive Ca²⁺ release from ER. The TG-sensitive ER Ca²⁺ stores can be displayed as ΔF350/F385 (b). After ER Ca²⁺ stores are completely depleted (~900 s), addition of extracellular 2 mM Ca²⁺ (indicated by second arrow) activates a sustained intracellular Ca²⁺ elevation through SOCE, which can be represented by ΔF350/F385 (c). This SOCE can be blocked by SOCE inhibitor 2-APB (indicated by third arrow)

3.4. Mn²⁺ Quenching Recording and Analysis

1. Isosbestic point, a specific wavelength at which the total fluorescence of Fura-2 does not change along with Ca²⁺ concentration, should be determined first. Perform excitation wavelength scanning of Fura-2 in solutions containing various Ca²⁺ concentrations ranging from 0 to 39.8 μM to determine the Ca²⁺ concentration-independent wavelength as the isosbestic point. Generally, the isosbestic point is at 360 nm, but may be slightly different in different optical system (see Note ⁷).

2. Mount the dish with C2C12 myotubes on the stage of the microscope, and select the “excitation ratio” mode with excitation wavelengths at 360 nm and 385 nm and emission at 510 nm. Record the fluorescence intensity at 510 nm with excitation wavelengths at 360 and 385 nm. The ratio (F_{360 nm}/F_{385 nm}) can be displayed in separate windows, providing information regarding intracellular Ca²⁺ homeostasis: resting intra-cellular Ca²⁺, TG-releasable ER Ca²⁺ stores, and SOCE (see Note ⁸).

3. Pretreat the C2C12 myotubes with 20 μM BTS (a selective inhibitor of the ATPase activity of skeletal muscle myosin II that reversibly blocks gliding motility and suppresses force and twitch production in fast skeletal muscle) for 15 min in BSS-Ca solution.

4. Select ROIs for individual myotubes.

5. Perform the running protocol: BSS-Mn (30 s), BSS-TG/Mn (900 s), or BSS-TG/Mn-2APB (300 s) and Triton X-100 (Fig. 2a) (see Note ⁹).

6. The decay of Fura-2 fluorescence upon Mn²⁺ addition is expressed as percentage decrease in Fura-2 fluorescence per unit time (the initial fluorescence is set to be equal to 100%). The completely quenched fluorescence signal is established at the end of each experiment by lysing the cells with 0.1% Triton X-100 (as 0%).

7. F360 curve is fitted into sigmoidal curve using Origin Pro7.5 (OriginLab, MA). To quantify the graded activation of SOCE, the first-order derivative of changes in sigmoidal curve of F360, dF360/dt, is analyzed [6] (Fig. 2b). This analysis provides two useful kinetic parameters: SOCE_max (d), the peak slope of Mn²⁺ quenching, and Δτ, the duration from initiation of TG-induced Ca²⁺ release (t₀) to the point where SOCE_max is reached (t₁).

Fig. 2.

Mn²⁺ quenching assay reveals graded activation of SOCE in C2C12 myotubes. (a) The decay slope of Fura-2 fluorescence is measured at the Ca²⁺-independent excitation wavelength 360 nm of Fura-2 (F₃₆₀). Addition of Mn²⁺ only (indicated by first black arrow) is recorded as baseline; the completely quenched fluorescence signal is established at the end of each experiment by lysing the cells with 0.1% Triton X-100 (as 0%). Simultaneous application of TG and Mn²⁺ (second black arrow, t₀) results in a gradually accelerated decay of fluorescence until reaching a maximal quenching rate. The gradual developed Mn²⁺ quenching can be extracted for further analysis (between dashed blue lines). (b) The extracted trace of F₃₆₀ can be fitted in a sigmoidal curve (red line). The activation of SOCE is expressed as percentage decrease in fluorescence per unit time (the initial value as 100%). To quantify the graded activation of SOCE, the first-order derivative of changes, dF360/dt, is determined (blue dotted curve). This analysis provides two useful kinetic parameters: SOCE (d), the peak slope of Mn²⁺ quenching, and Δτ, the duration from initiation of TG-induced Ca²⁺ release (t₀) to the point where SOCE_max is reached (t₁)