Ds qi2 camera

To assess bacterial viability, the tested samples were dispersed in LB or TSB growth medium for 30 s (A = 18%, W = 250 W, on:off = 2:1 s) and mixed with bacteria to a final volume of 1 ml (OD₅₅₀ = 0.3). After incubation for 12 h, 100 μl was centrifuged for 5 min at 6000 rpm, and the supernatant was replaced with 25 μl of Live/Dead BacLight Bacterial Viability Test (Invitrogen, Thermo Fisher Scientific) containing SYTO9 and propidium iodide (PI) in 1X phosphate-buffered saline (PBS) at a 1:1 ratio and a concentration of 3 × 10⁻⁶ mg/ml. It was followed by a 15-min incubation in the dark to stain the bacteria. Fluorescent bacteria were visualized by a Nikon inverted fluorescence microscope ECLIPSE Ti-S/L100 (Nikon) coupled with a DS-Qi2 Nikon camera (Nikon).

Morphological characterization of planktonic cells and biofilms grown in the four media was performed. To this end, ON cultures were grown in the four culture media at 30°C, the standard temperature for yeast growth in laboratory settings, and at 37°C, the natural body temperature. Moreover, the effect of FBS 10% supplementation in planktonic cultures incubated at 37°C was also tested. Cells were recovered and washed twice with PBS (4000 rpm x 5 min). PBS-resuspended yeasts were imaged under a 100x magnification with a Nikon inverted fluorescent microscope ECLIPSE Ti–S/L100 (Nikon, Japan) coupled with a DS-Qi2 Nikon camera (Nikon, Japan) in Bright Field.
On the other hand, C. parapsilosis biofilms formed on the fed-batch condition (ALI and Bottom zone) and the continuous flow system were visualized via chitin staining with 10 µM Calcofluor white (CW) (Biotium, USA) under a Zeiss LSM 800 confocal scanning laser microscope (CSLM). Images were processed and measured using ImageJ Fiji software. Briefly, the area and the Length-to-Width Ratio (LWR, denoted as aspect ratio in ImageJ) were calculated from randomly chosen cells (n=30) from planktonic and biofilm conditions. Pseudohyphae were defined as forming chains of cells with box-shaped ends and LWR greater than three, and blastospores as cells oval-shaped with a lower LWR (Rupert and Rusche, 2022 (link)).

C2C12 cells were grown in 35-mm glass surface dishes (Fluorodish FD35, World Precision Instruments), differentiated for 3 days then transfected with Tug1 or control LNA (25 nM) for 24 h. Mitochondria were labelled for 30 min with 50 nM MitoTracker RedCMXros (M7512, Life Technologies), then fixed with 2% paraformaldehyde in PBS for 10 min at 37°C. Fixed cells were then stained for F-actin with phalloidin (1:1000 in PBS, A22287 Life Technologies) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) to visualise nuclei (1:1000 in PBS, #62248 Thermo Scientific). All control and Tug1 LNA cells were prepared and imaged in parallel under the same conditions. Confocal microscopy (Nikon Eclipse Ti2, Australia) was performed using a 100x CFI Plan Apo Lambda NA 1.45 oil-immersion objective and Nikon DS-Qi2 camera. Excitation/emission wavelengths were 561/595 nm (MitoTracker), 640/700 nm (phalloidin) and 405/450 nm (DAPI), respectively. Z-stack images (0.15 μm steps) were acquired using software (Nikon Elements v5.21.03). Mitochondrial morphology in each Z stack image was quantified in ImageJ (NIH, Bethesda, MD) using a macro described previously [65 (link)]. Morphology parameters were calculated as follows: aspect ratio = major axis/minor axis, roundness = 4 × ((area)/(π × major axis²).