μ slide 8 well high chambered coverslip

Primary ear fibroblast cells were plated in fibronectin-coated glass-bottom μ-Slide 8 Well high chambered coverslip (ibidi). 24 h after cell plating, the samples were taken for FCCS experiment. Two-color FCCS experiments were performed for both nuclear and cytoplasmic compartments in each cell. Each FCCS experiment was performed for 10 s with 3 technical repeats on the same spot. The experiments were performed using Zeiss LSM880 Airyscan/FCS confocal microscope system with environmental control. The mEGFP was excited with the 488-nm Argon laser line and the mScarlet was excited with the 561-nm DPSS laser line. The C-Apochromat, 40x, 1.2 NA water-immersion objective was used for all the experiments. The emission signal was split by a dichroic mirror MBS 488/561/633 and detected at 505–530 nm for mEGFP and 570–600 nm for mScarlet using the GaAsP PMT array detector. The confocal pinhole was set for 1.0 airy unit (approximately 0.8756 and 1.33 fL for 488 nm and 561 nm) for both channels calibrated in the x-y plane for maximum signal intensity.