Fluorodish tissue culture dishes

50,000 VM-M3 cells were plated in 35 mm Poly-D-Lysine coated glass FluoroDish tissue culture dishes (World Precision Instruments) in 1 mL control media and allowed to grow for 24 hours. At 24 hrs, cell were administered treatment: media was replaced with high glucose media (control; 25mM), low glucose media (LG; 3mM), control media with ketone supplementation (βHB; 5mM βHB), control media with a single session of hyperbaric oxygen (HBOT; 100% O2, 90 min, 2.5 ATA), or combination therapy (LG+βHB+HBOT). At 48 hours, cells were incubated in 10μM DHE (Ex/Em: in CSF for 30 min at 37°C, and superoxide production was measured using fluorescence confocal microscopy with a TRIT-C filter as previously described [56 (link)]. Treatments were run in replicate. Superoxide production was measured by calculating the average of the fluorescence intensity units (FIU) per cell in 10 frames per sample using a 10x objective.

MEFs were isolated from day 13.5 C57Bl/6 mouse embryos and cultured as previously described [16 (link)]. Cultured cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) foetal bovine serum (FBS) at 37°C, 5% CO₂. For imaging experiments, MEFs at passages 1–3 were seeded into Fluorodish^™ tissue culture dishes (World Precision Instruments Pty Ltd) and grown to 70–90% confluency. Cell transfections were performed using Lipofectamine 3000 reagent (Life Technologies) and plasmid DNA according to the manufacturer’s instructions. Jasplakinolide (Sapphire Bioscience Pty Ltd) was added at a final concentration of 7 μM from a 1mM stock prepared in DMSO. FRAP analysis was performed within 10 s after addition of the drug.

Diseased, surgically resected palmar fascia was disaggregated as previously described,²³ (link) and myofibroblasts were seeded on 35 mm FluoroDish tissue culture dishes (World Precision Instruments) at 50,000 cells per dish. Cells were fixed in 4% formaldehyde and permeabilized using 0.1% triton X. SFRP4 was stained using goat anti-SFRP4 primary IgG antibody (AF1827, R&D Systems) and rabbit anti-goat IgG Alexa Fluor 633 (A-21086, Thermofisher Scientific). Filamentous actin and nuclei were stained using Acti-stain 488 phalloidin (Cytoskeleton Inc.) and Hoechst 33342 (Thermofisher Scientific). Fluorescence images were acquired using a confocal laser scanning microscope (Zeiss LSM 710) with a 40× objective.