Cellstar μclear 96 well tissue culture plates

HEK293 cells stable expressing CLuc-FGF14 and CD4-Nav1.6-NLuc (Clone-V) were grown for 24–48 h. Clone-V cells were detached using TrypLE (Gibco, Waltman, MA, United States), triturated in medium, and seeded in white, clear-bottom CELLSTAR μClear® 96-well tissue culture plates (Greiner Bio-One) at ∼0.8 × 105 cells per well in 200 μl of medium. The cells were treated for 12 h in a growth medium supplemented with 100 μl of serum-free, phenol red–free DMEM/F12 medium (Invitrogen) containing PLEV or EYYV (1–250 μM). The final concentration of DMSO was maintained at 0.5% for all wells. Following 12 h incubation at 37°C, the luminescence reaction was initiated by injection of 100 μl substrate solution containing 1.5 mg/ml of D-luciferin dissolved in PBS (final concentration = 0.75 mg/ml) by the Synergy™ H4 Multi-Mode Microplate Reader (BioTek). LCA readings were performed at 2 min intervals for 20–30 min, integration time 0.5 s, while cells were maintained at 37°C throughout the measurements. Detailed LCA method can be found in previous studies (Shavkunov et al., 2012 (link); Ali et al., 2014 (link), 2016 (link), 2018 (link); Hsu et al., 2015 (link); Wadsworth et al., 2019 (link), 2020 (link); Singh et al., 2020 (link)).

Cells were trypsinized (0.25%), triturated in medium, and seeded in white, clear-bottom CELLSTAR μClear^® 96-well tissue culture plates (Greiner Bio-One, Monroe, NC, USA) at ~ 0.9 × 10⁵ cells per well in 200 μL of medium. For transiently transfected cells, the trypsinization occurred 48 h post-transfection. The cells were incubated for 24 h, and the growth medium was subsequently replaced with 100 μL of serum-free, phenol red–free DMEM/F12 medium (Invitrogen) containing inhibitors (0.25–50 μM). The final concentration of DMSO was maintained at 0.3% for all wells. Following 2 h incubation at 37 °C, the reporter reaction was initiated by injection of 100 μL substrate solution containing 1.5 mg/mL of D-luciferin dissolved in PBS (final concentration = 0.75 mg/mL) by the Synergy^™ H4 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Luminescence readings were performed at 2-min intervals for 20 min, integration time 0.5 s, and the cells were maintained at 37 °C throughout the measurements. Signal intensity for each well was calculated as a mean value of peak luminescence; the calculated values were expressed as percentage of mean signal intensity of the per plate control samples.