Fura 2 am

HEK293T and DRG cells were loaded with 100 nM Fura-2 AM (Sigma) for 40 minutes before washing with extracellular fluid (in mM: NaCl 150, KCl 5, MgCl₂ 2, CaCl₂ 2, HEPES 10, glucose 10; pH 7.4). Coverslips were mounted on a Nikon Eclipse TE300 microscope with an optiMOS Scientific CMOS Camera (Teledyne QImaging, Birmingham, United Kingdom), and images acquired with Winfluor (Version 3.7.3; University of Strathclyde, Glasgow, United Kingdom) every 2 seconds with excitation at 355 and 380 nm and emission recorded at 510 nm. Control buffer and drugs were perfused via a 6-way gravity flow system (∼600 μL/minute). The outflow was placed ∼100 to 200 μm from the group of cells from which the recordings were made. Recordings were performed at 37°C, except where the effect of heat stimulation was assessed when the temperature of the superfusate was increased from 32 to 55°C linearly. The temperature of the superfusate was continuously measured at the opening of the superfusing tube and recorded alongside measuring fluorescent intensities.

The method described previously (Chan et al., 1996 (link)) was used in present study. Briefly, cerebral cortex was digested with 0.25% trypsin (Sigma, USA) at 37°C for 20 min. The reaction was terminated with DMEM containing 10% FBS (Hyclon, USA). Samples were filtered through a cell strainer, and centrifuged at 1,000 rpm for 5 min. The precipitated cells were resuspended in D-Hank's solution and adjusted to 1 × 10⁶ cells/ml. They were loaded with 5 μM fura-2 AM (Sigma, USA) at 37°Cfor 30 min in the dark, washed twice with D-Hank's solution, and incubated at 37°C for 5 min in the dark. Concentrations of intracellular free Ca²⁺ [Ca²⁺]i were determined by using fluorospectrophotometer (Hitachi F-4500, Japan). The following equation was used for calculation:
[Ca²⁺]i = Kd × [(R–Rmin)/(Rmax–R)] × (Sf380/Sb380), where Kd is the dissociation constant of the dye (224 nM was used); R is the ratio at excitation wavelengths 340/380 nm; Rmin is the ratio at zero [Ca²⁺]i, and Rmax is the ratio at saturated [Ca²⁺]i. Rmax was obtained by adding 0.2% Triton X-100 to make cell membrane permeable to Ca²⁺, allowing the extracellular and intracellular free Ca²⁺ to equilibrate. Thereafter, Rmin was determined by adding chelator to erase all extracellular and intracellular free Ca²⁺. Results were expressed as nmol/L (10⁶ cells/ml).

Monitoring of cytosolic calcium transients in individual neurons was carried out using the membrane permeable fluorescent indicator Fura 2-AM (Sigma-Aldrich) in combination with the Till Vision Imaging System (T.I.L.L. Photonics, Gräfelfing, Germany) coupled to an upright microscope (Axioskop 2 FS plus, Zeiss). Emitted fluorescence was collected by a charge-coupled device (CCD) camera. Cultured cells were incubated for 30 minutes at 37°C with Fura 2-AM in a standard bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM glucose and 10 mM HEPES, adjusted to pH 7.4 with NaOH.
The intracellular Ca²⁺ was imaged by exciting Fura 2-AM at 340 and 380 nm with its emission monitored in intervals of 300 ms at 510 nm. Recordings were terminated by a 50 mM KCl stimulation to ensure the viability of the recorded cells. After background subtraction, the 340/380 nm excitation ratio for Fura 2-AM was calculated, which increases as a function of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). To determine [Ca²⁺]_i a calibration measurement in the presence of 5 μM ionomycin or with a 10 mM EGTA solution free of Ca²⁺ was conducted. [Ca²⁺]_i was calculated according to [Ca²⁺]_i = β × K_D(R ‒ R_min)/(R_max ‒ R) [55 (link)] with β = F_380,max/F_380,min = 3.6, K_D = 245 nM, R_min = 0.38 and R_max = 1.6.

Cells were plated on a 24-well plate at the density of 4 × 10⁴. After treatment, the cells were washed with 1 mL of serum-free medium and loaded with 20 µM FURA2 AM; (Sigma-Aldrich, USA) in the presence of 0.5% pluronate (Sigma-Aldrich, USA) and 0.1 nM ionomycine in serum-free medium for 40 min at 37 °C in the dark. The cells were then washed three times with a 500 µL of PBS. Fluorescence was measured on the fluorescence scanner Synergy II (BioTek, Germany) at λex 340/380 nm and λem 516 nm. The results were calculated as the ratio between 340 and 380 nm and expressed as relative fluorescence units (RFU).

FURA-2AM (FURA-2-pentakis (acetoxymethyl) ester), PBS (Phosphate-Buffered Saline), Triton X-100, EGTA (ethylene glycol-bis (β-aminoethyl ether), sodium elenite (Na₂SeO₄), selenium methionine, hydrogen peroxide (H₂O₂), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl₂), glucose, Hepes, and dimethyl sulfoxide (DMSO), were acquired from Sigma-Aldrich (St. Louis, MO, USA). Any other chemicals and reagents (reagent grade) were of the highest quality, and obtained from reputable commercial sources.