μ plate 96 well ibitreat

Prepared cells were plated on 96-well polymer coverslip bottom plates (μ-Plate 96-Well ibiTreat; ibidi). R-GECO1 fluorescence imaging with blue light illumination for OptoSTIM1 activation was performed using a Nikon A1R confocal microscope (Nikon Instruments), mounted onto an inverted Eclipse Ti body (Nikon), equipped with a CFI Plan Apochromat VC objective (× 60 /1.4-numerical aperture (NA)) and digital zooming Nikon imaging software (NIS elements AR 64-bit version 3.21; Laboratory Imaging). During image processing, cells were maintained at 10% CO₂ and 37 °C by incubating in a Chamlide TC system (Live Cell Instruments, Inc., Korea). Immediately before imaging, the medium was replaced with OPTI-MEM (Invitrogen). Blue light photo-excitation (power density, 500 μW mm⁻²) was delivered with a 488-nm laser at 5-second intervals for 1 minute, unless stated otherwise.

HeLa cells (obtained from American Type Culture Collection) were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in a humidified 10% CO₂ atmosphere. Cells were confirmed to be free from mycoplasma using an e-Myco Mycoplasma PCR detection kit (iNtRON). For live-cell imaging, cells were plated on a 96-well plate (μ-Plate 96 Well ibiTreat; ibidi GmbH) and transfected using either a Microporator (Neon Transfection System; Invitrogen) or Lipofectamine LTX (Invitrogen), according to the manufacturers’ instructions. For HeLa cells, electroporation conditions (two pulses of 980 V for 35 ms) were additionally optimized for higher transfection efficiency.