Nis element ar 64 bit version 3

HeLa cells were transfected using a Microporator (Neon Transfection System; Invitrogen) in a condition of two pulses of electric shock at 980 V for 35 milliseconds. At 12 hours post transfection, cells were treated for 2 hours with dimethyl sulfoxide (DMSO) or Rapamycin (Calbiochem), dissolved in DMSO as 2 mm stock solution and diluted to 500 nM in DMEM before use. To induce oligomerization of CRY2-fused proteins, cells were exposed to pulsatile illumination of light (5-s irradiation every 10-s, 470 nm, 100 μW mm⁻²) administered with a blue LED array for 30 minutes before fixation. Cells were then fixed with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) for 20 minutes and washed with PBS for three times. Cells were imaged using a confocal microscope and analyzed with NIS-element AR 64-bit version 3.21; Laboratory Imaging software provided from Nikon.

For live-cell imaging, a Nikon A1R confocal microscope (Nikon Instruments), mounted onto a Nikon Eclipse Ti body and equipped with a CFI Plan Apochromat VC objective (× 60/1.4-numerical aperture (NA)) and digital zoom Nikon imaging software (NIS Element AR 64-bit version 3.21; Laboratory Imaging), was used. Cells were maintained at 5% CO₂ and 37 °C during imaging with the microscope-mounted Chamlide TC System (Live Cell Instruments, Inc., Korea). Ca²⁺ influx in cells co-expressing R-GECO1 was measured using genetically encoded red Ca²⁺ indicators with each of the monSTIM1 variants; 488 and 561 nm laser sources were used to excite cells at 30-s intervals. Captured images were analyzed using NIS-Elements AR microscope imaging software (NIS-element AR 64-bit version 3.21; Nikon). Time-lapse images of Ca²⁺ influx were analyzed in defined regions of interest (ROI) in the cells, and changes in R-GECO1 intensity were quantified.