Axiovert 100 tv epifluorescence microscope

The A549 cells were grown in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1% penicillin/streptomycin (Sigma Aldrich). The A549 cells were seeded and grown to 70% confluency on an 8-well chambered coverglass (Lab-Tek, Nunc, USA). The cells were incubated with 1 μM probe in media for 30 min at 37℃. For blocking, 200 μM excess cRGD was added to the cells for 5 min prior to probe treatment and remained present during probe incubation. The cells were washed three times with 1xPBS, fixed with 4% cold paraformaldehyde for 20 min at room temperature, washed again with 1xPBS, and co-stained with 3 μM Hoechst 33342 for 10 min. Afterwards, the cells were washed two times with 1xPBS and imaged on a Zeiss Axiovert 100 TV epifluorescence microscope equipped with UV filter (Ex. 387/11, Em. 447/60) and Cy5.5 filter (Ex. 655/40, Em. 716/40). For each micrograph, a background subtraction with a rolling ball radius of 200 pixels was conducted using ImageJ2 software. The average mean fluorescence intensity for each micrograph was then calculated from using 20 randomly generated 25 × 25 pixel extra-nuclear ROIs. The averages and SEM were calculated and plotted in GraphPad Prism.

All microscopy images were collected with a Zeiss Axiovert 100 TV epifluorescence microscope equipped with a UV filter (ex: 387/11, em: 447/60), TxRed filter (ex: 562/40, em: 624/40), and Cy5.5 filter (ex: 655/40, em: 716/40).

HT-1080 cells were seeded into 8-well chambered slides (Lab-Tek, Nunc, USA) and were grown to 70% confluency. The cells were incubated with 1 μM SF8 probe in media for 30 min at 37 °C. The cells were washed three times with phosphate buffered saline and co-stained with either 100 nM MitoTracker Green FM (Thermo Fisher Scientific) or 100 nM LysoTracker Red DND-99 (Thermo Fisher Scientific) for 15 min in opti-MEM at 37 °C. Cells were then washed two times with phosphate buffered saline and incubated with 3 μM Hoechst 33342 for 10 min at room temperature. The live cells were washed two additional times and imaged on a Zeiss Axiovert 100 TV epifluorescence microscope equipped with a UV filter (ex: 387/11, em: 447/60), FITC filter (ex: 485/20, em: 524/24), TxRed filter (ex: 562/40, em: 624/40), and Cy5.5 filter (ex: 655/40, em: 716/40). Image processing for each micrograph was then conducted using ImageJ2 software with a 10-pixel rolling ball radius background subtraction.