Lsm 780 laser scanning confocal microscope

The effect of LL-37 on plasma membrane integrity of C. auris was examined by Propidium Iodide (Sigma-Aldrich) staining method as it is used as a universal marker for studying plasma membrane permeability [28 (link)]. The experiment was conducted as previously described [29 (link)], with modifications. Briefly, C. auris MRL6057 cells were inoculated in SDB and incubated at 37 °C, 200 rpm for 24 h. Post-incubation cells were washed twice with sterile PBS, resuspended in SDB (turbidity was adjusted to 0.5 McFarland), and exposed to an appropriate concentration of LL-37 (MIC and MFC) for 4 h. Both positive (exposed to H₂O₂, 10 mM) and negative controls were included in the study. The cells were then washed twice with PBS and stained with PI (30 µM), and incubated for 30 min at room temperature in the dark. After incubation, cells were rewashed with PBS, and the pellet was resuspended in PBS (250 µM), and 10 µM of the sample was used for fluorescence microscopy (Zeiss Laser Scanning Confocal Microscope (LSM) 780 and Airyscan (Carl Zeiss, Inc. Jena, Germany).

The TUNEL assay, also referred to as terminal deoxy-nucleotidyl transferase dUTP nick end labelling staining, is used to identify DNA breaks or fragmentations produced during the last stages of apoptosis. For the TUNEL assay, overnight grown C. auris cells were collected and resuspended in PBS followed by treatment with ½MIC, MIC, and MFC values of the compound C5. H₂O₂ treated C. auris cells acts as the positive control and healthy cells serve as the negative control. The cells were rinsed three times with PBS and fixed in fixative solution (4% paraformaldehyde) for 30 minutes. The cells were put in 0.25% triton X-100 for 2 minutes for permeabilization. After permeabilization the C. auris cells were washed with PBS and incubated with Click-iT Plus TUNEL-assay kit (Thermo Fisher Scientific’s) for 30 minutes in the dark. Additionally, Candida cells were stained with 50μl of Hoechst 33342 (1X) dye (Thermo-Fisher Scientific USA) and left-over to sit for 15 minutes in the dark at room temperature, and cell were washed two time with PBS, before going to observe under the Confocal Microscopy (Zeiss Laser-Scanning Confocal-Microscope (LSM) 780 Jena, Germany). The excitation-wavelength of 495nm and an emission-wavelength of 519nm for the Click-iT Plus TUNEL assay dye and for Hoechst-33342 dye had an excitation wavelength of 350nm and an emission wavelength of 461nm was used.

The OVCAR-5 cell line was cultured on coverslips until reaching 70% confluency. Cells were then treated with MSN_CurNQ for 24 h, thereby allowing for cellular uptake to occur. After this, Phalloidin-Atto 488 was added, and staining occurred over 30 min. Cells were then washed and fixed through incubation in 5% formaldehyde in PBS for 20 min. After fixation, DAPI stain was added at a concentration of 1 μg/mL and samples were left to incubate for 15 min in the dark. Hereafter, samples were washed thoroughly with PBS to remove unstained DAPI. Coverslips were stored in the dark in PBS. Coverslips were viewed on the Zeiss Laser Scanning Confocal Microscope (LSM) 780 (Oberkochen, Germany). Super resolution microscopy was performed using Airyscan technology as an added on feature to the LSM confocal microscope.

The DNA FISH probes were synthesized by Empire Genomics Inc (for the detailed information about the chromosomal location and fluorescence labelling of the probes, see Supplementary Table 2). Human lymphoid cell line GM12878 cells were fixed and permeablized following the protocols3 (link)51 (link) with slight modification. The denaturation and hybridization steps were performed according to the protocols suggested by the manufacturer. Three probes (150 ng each) for either targeted regions or control regions were mixed thoroughly with 18 μl hybridization buffer (provided by the manufacturer) and applied evenly with the sample on a microscope slide. After hybridization, the samples were washed several times to remove unbound FISH probes, and mounted on microscope slides with 10 μl DAPI mounting solution for each slide. The FISH images were acquired with Zeiss Laser Scanning Confocal microscope (LSM-780) with × 63 oil immersion objective lenses. Optical sections (Z stacks) with 0.25–0.5 μm apart were obtained with alternative scanning (frame mode) of two lasers each, and stored in four separate channels with the ZEN software provided by the manufacturer. The FISH results were analysed with ImageJ52 (link) and Nemo53 (link).

In total, 8 × 10⁴ skin fibroblasts from each patient with PMM2 and age-matched control fibroblasts were seeded on chambers and fixed with 4% paraformaldehyde. Cells were blocked for 1 h in Blocker^TM Casein in PBS (Thermo Fischer). Then, cells were stained with rabbit anti-LC3 antibody (Novus Biologicals; Cat# NBP2-46892) overnight at 4 °C and visualized via incubation with anti-Rabbit IgG Alexa Flour^TM 488 (Thermo Fischer Scientific; Cat# A-21206) for 1 h at 4 °C. These fibroblasts were counterstained with DAPI (Vector Laboratories, Newark, CA, USA; Cat# H-1200). Images were acquired using Carl Zeiss laser scanning confocal microscope LSM780 and processed with Zen black software.

The intracellular accumulation of R6G was examined using the method previously described [33 (link)]. Briefly, C. auris MRL6057 was exposed, similarly to the above, resuspended in PBS supplemented with glucose (2%) and R6G (4 µM), and kept for 30 min at room temperature. After that, centrifugation was conducted, cells were mixed with PBS, and slides were made for analysis through fluorescence microscopy (Zeiss Laser Scanning Confocal Microscope (LSM) 780 and Airyscan) (Carl Zeiss, Inc.).