A1r confocal laser scanning microscope system

Fresh mycelia were collected for the live cell imaging by shaking mycelia for 24 h in CM. A Nikon A1R laser scanning confocal microscope system (Nikon, Japan) was used for the live-cell imaging. GFP excitation was performed with 488 nm light (Em. 525/40 nm). The hyphal tips were visualized by staining with FM4-64 at a final concentration of 10 mg/mL with 405 nm light (Em. 452/45 nm) and photographed.

Confocal images were recorded to assess the proportion of cell membrane expression of wildtype NET and mutants. The cell sample dish was mounted above a 60x oil immersion objective on a Nikon A1R+ laser scanning confocal microscope system (Nikon, Minato City, Tokyo, Japan). Images were acquired using a resonant scanner. The nisoxetine-based fluorescent probe AC1-146 and trypan blue solution were excited by a 561-nm laser line; mGFP was excited by a 488-nm laser line. A 525/50 nm and a 595/50 nm emission filter were used. The emitted light was collected with a high-sensitivity GaAsP detector. To assess NET internalization after PIP₂ depletion, the ratio between plasma membrane fluorescence (F_PM) and cytosolic fluorescence (F_Cyt) was determined with ImageJ software^{57 (link)}. The regions of interest (ROIs) were drawn in the 561-nm channel where the stained plasma membrane was clearly visible. Total fluorescence was calculated by multiplying the area and average fluorescence of each ROI in the 488-nm channel after background subtraction.

Images were taken with a Nikon A1R + laser scanning confocal microscope system with a 60× NA 1.4 oil immersion objective (Nikon, Vienna, Austria). After aspiration of cell culture medium, cells were incubated with trypan blue (0.4%, Sigma-Aldrich) for 10 min and subsequently washed with KHB. eYFP fluorescence was excited with a 488 nm laser line, trypan blue with a 561 nm laser and monomeric BFP with a 399 nm laser. A 525/50 nm, 595/50 nm or 454/50 nm emission filter was used for eYFP, trypan blue or mTagBFP, respectively. Images were analysed as previously described [38 (link)]. In brief, images, taken on at least three separate days, were analysed with Fiji ImageJ 1.53c. Membrane expression was assessed by drawing the cell membrane and the interior of the call and calculating relative expression at the membrane.

For morphological changes in treated and untreated cells, phase contrast microscopy was used. Confocal microscopy was used according to the methodology given by Alam et al. [19 ] with slight modifications for determination of nuclear changes. HepG2 cells were seeded in a 6 well plate at a concentration of 5 × 10⁵ cells per well for 24 h. Thereafter, the HepG2 cells were treated with IC₅₀ of herbal combination and the positive control, camptothecin. Following incubation for 24 h, the cells were washed thrice with phosphate buffered saline (0.1 M) and fixed with paraformaldehye (4%) for 30 min. Finally, the slides were prepared after staining the cells with 4′,6-diamidino-2-phenylindole (DAPI) (10 μg/ml) and Nikon A1R laser scanning confocal microscope system (Nikon Corp., Japan) fitted with Nikon 40X 0.95 NAD-ICM/N2 plan objectives was used to capture images. The acquisition of images and analysis was carried out using the built-in Nikon NIS Element AR software and the fluorescence was observed with a long-pass 488 emission filter.

Nocodazole (Sigma, final concentration 100 μM), LatA (latrunculin A, Sigma, final concentration 10 μM) and CFW (Calcofluor White, Sigma, final concentration 10 μg/ml) were used according to our previously reported [9 (link),12 (link)]. Nikon A1R laser scanning confocal microscope system was used for live cell fluorescence imaging (Nikon, Japan). Elapsed time is indicated in seconds. CFW excitation used 405 nm light (Em. 452/45 nm), GFP excitation was performed with 488 nm light (Em. 525/40 nm), HCS LipidTox Redor mCherry excitation was performed with 561 nm light (Em. 607/36 nm).

Confocal microscopy and image analysis was conducted as previously described [32 ]. In brief, cells expressing yellow fluorescent protein(YFP)-tagged human SERT or DAT, respectively, were seeded on PDL-coated 35 mm glass-bottom dishes 24 h before image acquisition. Culture medium was removed and the cells were exposed to the substances of interest (10 µM: S-4-MC, R-4-MC, S-4-MMC, S-4-TFMMC, R-4-TFMMC. 30 µM: R-4-MMC) for 1 h. Subsequently, trypan blue (0.4 %) was added for 10 min. Cells were washed with KHB and kept in buffer during image acquisition. A Nikon A1R+ laser scanning confocal microscope system with a ×60 NA 1.4 oil immersion objective (Nikon, Vienna, Austria) was used for imaging. Trypan blue was excited at 561 nm and the YFP-tagged transporter with a 488 nm laser line. Emitted light was filtered appropriately and detected with a GaAsP PMT detector. Four to five images were taken on 3 separate days. Fiji ImageJ 1.53c was used for image analysis. For each image, two regions of interest were hand-drawn per cell. One encompassed the cell membrane (defined by trypan blue staining) and one the cell interior. Membrane transporter expression levels were calculated as the ratio of membrane versus intracellular mean intensity.