Fv1200 spectral laser scanning confocal microscope

PCa cells were cultured in chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) under normoxia or hypoxia for 48 hrs. Thereafter, cells were fixed, permeabilized, and incubated overnight with primary antibodies (1:500; CD63, actin, and Rab5). Cells were then incubated with secondary anti-rabbit and/or anti-mouse antibodies conjugated with Alexa Fluor, followed by staining with Prolong® Gold Antifade Reagent with DAPI. Fluorescent images were captured using an Olympus FV1200 SPECTRAL Laser scanning Confocal Microscope at 60× objective lens (Olympus IX83 inverted platform).

The detection was performed mainly by combining RNA Fluorescence in situ Hybridization (FISH) (probe for Linc00942 is synthesized from LGC Science Ltd) and protein Immunofluorescence (IF) with some modifications. Firstly, cells were seeded in cell climbing sheets of a 12‐well plate and fixed using Fixation Buffer after well adhering, then incubated with the Stellaris RNA FISH probe (LGC) in Hybridization Buffer for at least 4 h at 37°C after using .2% Triton X‐100 for 20 min to permeate the cells. After washing with Wash buffer A, the cells were blocked with 3% BSA for 1 h at room temperature and incubated with anti‐p62/SQSTM1 or anti‐LC3B antibody at 4°C overnight. After washing with PBST three times, the cells were incubated with secondary fluorescent antibodies (the Alexa Fluor 488 goat antirabbit IgG (H+L) secondary antibody) for 1 h at room temperature. before proceeding to imaging. All images were visualized and filmed using an Olympus FV1200 SPECTRAL Laser scanning Confocal Microscope.

OxS and OxR CT-26 CRC cells were plated onto collagen-coated cover slips and incubated for 24 h with 0 or 100 µg/mL of VMWNPs in either FBS-free media. Following incubation, VMWNPs solutions were removed and cells were washed twice with cold PBS, fixed with 4% paraformaldehyde and then stained with Alexa-fluor-488 and 4′,6-diamidino-2-phenylimdole (DAPI). An Olympus FV1200 SPECTRAL Laser scanning Confocal Microscope (Olympus IX83 inverted platform) was used to collect images of the cells and VMWNPs. To visualize the fluorescence from VMWNPs in vivo, a freshly euthanized female Balb/C mouse was intraperitoneally injected with 100 µL or a 250 µg/mL VMWNPs solution and imaged using a Perkin Elmer Caliper in vivo imaging system (IVIS). Lamp excitation of 465 nm was used and the indocyanine green (ICG) filter was used to capture emitted light above 695 nm.

MFL2 cells were infected with pBMN-mCherry-Parkin, which was a gift from Richard Youle (Addgene plasmid #59419) and seeded on polylysine (Sigma Aldrich) covered four-well chamber slides. Then the cells were treated with 100 µM devimistat or 10 µM FCCP for 24 h. Cells were incubated with MitoTracker Deep Red (ThermoFisher, Waltham, MA) for 30 min at 37 °C. After washing with PBS, the cells were fixed with 10% formalin at room temperature for 20 min. After washing twice with PBS, cells were incubated with VECTASHIELD^® mounting media with DAPI to stain the nucleus. Confocal microscopy was performed using an Olympus FV1200 SPECTRAL Laser scanning confocal microscope. All images were captured using the ×60 objective.

Cells were seeded on 8-well coverslip-bottom chamber slides at a density of 30,000 cells per well in 400 μL of complete medium for microscopy experiments, or on 6-well plates at a density of 500,000 cells in 3 mL of complete medium for flow cytometry experiments. Cells were allowed to adhere for 48 h and were then exposed to AgNPs for 24 h at 37 °C. For microscopy experiments, medium was removed and 10 µM Liperfluo dye (Dojindo Molecular Technologies, Rockville, MD, USA) diluted in 500 µL live cell imaging solution (Molecular Probes, Eugene, OR, USA) was added to each well. After 30 min Liperfluo-containing media was replaced with fresh live cell imaging solution and re-placed in incubator for 4 h. Fluorescence was then observed using an Olympus FV1200 spectral laser scanning confocal microscope. Fluorescence was quantified using ImageJ software. For flow cytometry experiments, medium was removed and 10 µM Liperfluo dye (Dojindo Molecular Technologies) diluted in 1 mL PBS was added to each well for 30 min. Cells were then washed with PBS, trypsinized, trypsin neutralized with complete media, and resuspended in fresh PBS. Cell fluorescence was measured within an hour of staining using a BD FACS Canto II Analyzer. Data analysis was performed using FCS Express 7 software (De Novo Software, Pasadena, CA, USA).