Attofluor cell chamber

HeLa cells (American Tissue Culture Collection: Manassas, VA, USA) and N1E-115 Neuroblastoma cells (European Collection of Cell Cultures: Salisbury, UK) were cultured using Dulbecco’s Modified Eagle Medium (DMEM) supplied with Glutamax, 10% FBS, Penicillin (100 U/ml) and Streptomycin (100 μg/ml). All cell culture media were obtained from Invitrogen (Bleiswijk, NL).
Cells were transfected in a 35 mm dish holding a glass 24 mm Ø #1 coverslip (Menzel-Gläser, Braunschweig, Germany), using 1–2 μl Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen), 0.5–1 μg plasmid DNA and 50 μl OptiMeM (Life Technologies, Bleiswijk, NL). N1E-115 cells were transfected in OptiMeM to accomplish neurite outgrowth by serum starvation53 (link).
Samples were imaged 1 day after transfection unless stated otherwise. After overnight incubation at 37 °C and 5% CO₂, coverslips were mounted in an Attofluor cell chamber (Invitrogen, Breda, NL) and submerged in microscopy medium (20 mM HEPES (PH = 7.4), 137 mM NaCL, 5.4 mM KCL, 1.8 mM CaCL₂, 0.8 mM MgCl₂ and 20 mM glucose). All live cell microscopy was done at 37 °C.

To investigate the contribution of Orai1 activity to scratch-induced changes in cytosolic Ca²⁺ and Na⁺, PAECs were grown on a 25-mm diameter coverglass (catalog no. 12-545-102, Fisherbrand) that was suspended in a 35 mm dish. Once the cells grew to confluence after 4–5 days, the monolayer was rinsed three times with Krebs-Henseleit (Krebs) buffer (catalog no. K3753, Sigma, ~156 mM Na⁺, 290 mOsm, pH 7.4) containing 2 mM CaCl₂ (catalog no. 223506, Sigma) and 25 mM HEPES (catalog no. 3375, Sigma). The monolayer was incubated at 37°C for 30 minutes with 6.315 μM Cal-590 AM (catalog no. 20510, AAT Bioquest) or 9.225 μM Ion NaTRIUM Green-2 AM (catalog no. ab142802, Abcam) in 1 mL Krebs buffer containing 8 μL Pluronic F-127 (catalog no. P6866, Life Technologies). The monolayer was then washed twice and incubated with 10 μM GSK-7975A or vehicle control (DMSO 1:1371 dilution) in 1 mL Krebs buffer at room temperature for 15 minutes. Finally, the coverglass was assembled into an Attofluor cell chamber (catalog no. A7816, Invitrogen), and 2 mL fresh 10 μM GSK-7975A or vehicle control was added. Scratching was done immediately before time-lapse imaging.

A suspension of f-ROS with fluorescent probe was introduced into a small chamber made of Secure-Seal from GRACE Bio-Labs (Bend, OR) on a glass slide (Matsunami, Tokyo, Japan) placed in an Attofluor Cell Chamber from Invitrogen (Paisley, UK). After 10 min, sedimentation of f-ROS onto the glass surface was complete. The chamber was set on the stage of a total internal reflection fluorescence microscope (TIRFM; Nikon, Tokyo, Japan, TE2000), and the aqueous phase was continuously perfused (0.1 ml min⁻¹) with a buffer containing an oxygen-scavenging system freshly prepared by mixing substrate (2.25 mg·ml⁻¹ glucose) and enzymes (216 µg ml⁻¹ glucose oxidase and 36 µg ml⁻¹ catalase). When GTP was applied to f-ROSs, the chamber was perfused with freshly prepared perfusion buffer containing 500 µM of GTP. Fluorescently labelled Fab′1D4 on disc membranes was illuminated with the highly inclined laser beam of 750-nm wavelength. Images were acquired with an electron-multiplying CCD camera C9100-12 (Hamamatsu Photonics, Hamamatsu, Japan) at a spatiotemporal resolution of 30 frames s⁻¹ and 76 nm pixel⁻¹. The TIRFM was equipped with Nikon 100×/1.45 Plan-Apo objectives. A filter set consisting of 760DRLP (Chroma Technology Corp., Bellows Falls, VT) and HQ810/90 (Omega Optical) was used.

Cell migration was observed using the ring-barrier migration assay as previously described [40 (link)]. Briefly, 3 × 10⁵ cells were seeded on fibronectin (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA)-coated coverslips in an Attofluor Cell Chamber (Invitrogen, Carlsbad, CA, USA) and migration was monitored for 24 h in DMEM medium supplemented with 1% FBS. Time-lapse imaging was conducted on an Axiovert 100 M inverted microscope (Carl Zeiss B.V., Sliedrecht, Netherlands). Parameters of cell migration were obtained from the acquired sequence using AxioVision SE64 Rel.4.9.1 software (Carl Zeiss, Oberkochen, Germany). Three independent experiments were performed in triplicates, and from each replicate, 10 cells’ trajectories were obtained.