Gcamp6 medium

N2a cells were plated on non-coated glass-bottom 35-mm dishes. Cells were cultured in DMEM supplemented medium. After 20-24 hours, cells were transfected using the Lipofectamine LTX Plus reagent (ThermoFisher #15338030) with GCaMP6 (GCaMP6 medium, Addgene cat.40754) and FeRIC channels as described above. Ca²⁺ imaging experiments were conducted at 20-24 h post-transfection.

N2a cells were plated on non-coated glass-bottom 35-mm dishes. Cells were cultured in DMEM supplemented medium. After 18-24 hours, cells were transfected using the Lipofectamine LTX Plus reagent (ThermoFisher #15338030) with GCaMP6 (GCaMP6 medium, Addgene cat.40754) and TRPV4^FeRIC channels. For transfection, OptiMEM free serum medium (ThermoFisher #31985088) was used to prepare the DNA/Lipofectamine LTX mix. Transfection mix had the following composition per each 35-mm dish: 300 μL OptiMEM, 4 μL Lipofectamine LTX, 3 μL PLUS reagent, 0.7 μg TRPV4 DNA, and 0.7 μg of GCaMP6 DNA. Where indicated, EGTA (pH 8, ThermoFisher Scientific, #NC0997810) was added to dishes after transfection to achieve 1 mM final concentration. It was estimated that 1 mM of EGTA decreases the Ca²⁺ concentration in the DMEM culture medium from ∼1.8 to ∼0.8 mM (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-TS.htm).

N2a cells were plated on non-coated glass-bottom 35-mm dishes at very low density (1 x10E5 cells). Cells were cultured in DMEM supplemented medium. After 20-24 hours, cells were transfected using the Lipofectamine LTX Plus reagent (ThermoFisher #15338030) with GCaMP6 (GCaMP6 medium, Addgene cat.40754) and TRPV4^FeRIC as described above. At 24-h post-transfection, the culture medium was replaced with fresh culture medium supplemented with HTF (500 μg/mL). Ca²⁺ imaging experiments were conducted at 72-h post-transfection.