Nis element c software

HEK293T cells co-transfected with pEGFP-C1, and either pRNAe-mock or pRNAe-egfpc1 plasmid was cultured for 6 h after transfection. Living cell fluorescence images were taken using a Nikon Ti-E inverted microscope (Nikon, Japan) every 10 min for 19 h. Relative average fluorescence strength was calculated using the NIS-Element C software (Nikon, Japan).

Dissected tracheae were treated sequentially with 0.1% Triton in PBS for 10 min, 0.1M ammonium chloride in PBS for 10 min, 50 mg/μl RNAse A (Thermo scientific) in PBS for 1 hour and finally DAPI (4', 6-diamidino-2-phenylindole, 0.01 mg/ml in PBS, Invitrogen) for 5 min. Tracheae were then detached from the cuticle for mounting. To quantitate DAPI intensity, image series were taken via a Zeiss LSM 710 confocal microscope with a 40x oil immersion objective. Nikon NIS-Element C software was then used to build 3D images of nuclei and quantitate DAPI intensity for each selected nucleus. An average of six nuclei were analyzed in each tracheal segment, and tracheae from eight individual larvae were analyzed per genotype. To quantitate cell numbers in Tr9 and Tr10 of the tracheal system, a Zeiss Axioplan2 was used, and 10 individual tracheal pairs were analyzed for each genotype.

SMCs were prepared for live-cell imaging prior to being placed in an incubation chamber (Chamlide TC; Live Cell Instrument, Seoul, Korea) kept at 37 °C and 5% CO₂. Briefly, the cells were cultured in a 12-well dish containing coverslips (diameter = 12 mm, Marienfeld, Germany) coated with 0.01% poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA). The cells were stained with 5 μM DRAQ5 (1,5-bis{[2-(di-methylamino) ethyl] amino}-4,8-dihydroxyanthracene-9,10-dione, ab108410, Abcam, Cambridge, UK), a cell permeable far-red fluorescent DNA dye, to track single-cell migration. At the same time, the cells were stimulated with or without flagellin (100 ng/mL). Confocal images were obtained with a Nikon A1R confocal microscope for 4 h using a 60× Plan Apochromat VC objective (NA 1.40) under illumination with a 633 nm laser. All images were processed, and quantification for single-cell tracking was performed using Nikon NIS Element C software (version 3.10).