Chromate-Induced Transmembrane Potential Imaging

The HEK293H cell line was obtained from ThermoFisher Scientific Invitrogen (Waltham, MA) and cultured with a DMEM/high glucose medium (GE Healthcare Life sciences, Piscataway) supplemented with fetal bovine serum 10% (ThermoFisher Scientific Gibco, Waltham, MA) in humidified incubator with 5% CO₂ at 37 °C. Cells were seeded at a density of 1 × 10⁵ per well. To record the transmembrane potential, cells were placed on a cover glass treated with poly-L-lysine 0.01% (Sigma-Aldrich). For the SICM imaging, cells were seeded on a polydimethylsiloxane (PDMS) substrate in a petri dish with 2 mm of thickness. The PDMS substrate was coated with collagen hydrogel matrix (5 μg/cm²) to improve the cell adhesion.^{29 (link)} If not mentioned otherwise, the HEK293H cells were treated with 10 μM K₂CrO₄ in extracellular solution for 90 min. For the fixed cell experiments by SICM, two batches of chromate treated cells were fixed with 4% paraformaldehyde for 10 min. Before patch-clamp or SICM experiments, the treated cells were rinsed two times with the extracellular solution.