The growth media for human fibroblasts were changed to DMEM FluoroBrite supplemented with 10% FBS 30 min before imaging. Cells were imaged using a TIRFM setup under a temperature-controlled environment (Tokai Hit) maintained at 37°C. Images were captured using Photometrics Prime 95B camera mounted on a Nikon Ti-2 microscope using Apo-TIRF 100×/1.49 oil-immersion or Apo-TIRF 60×/1.49 oil-immersion objective. Image acquisition was performed using NIS Elements Advanced Research Imaging software (version 4.60.00 [Build 1171] Patch 02). Images were processed and analyzed with NIS Elements and Matlab. Vesicle fusion events were analyzed by overlaying the time series images to determine all spots and by creating regions of interest around them as described previously (Ahmed et al., 2018 (link)). The fluorescence trace over time was extracted and analyzed using Matlab to determine bona-fide fusion events.
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Visualizing Fibroblast Vesicle Fusion Events
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Corresponding Organization : Victorian Clinical Genetics Services
Other organizations : Children's Hospital at Westmead, Royal Prince Alfred Hospital, Garvan Institute of Medical Research, St Vincent's Clinic, UNSW Sydney, Children's Cancer Institute Australia, Meyer Children's Hospital, University of Florence, University of Sydney
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Variable analysis
independent variables
- Growth media for human fibroblasts changed to DMEM FluoroBrite supplemented with 10% FBS
- Vesicle fusion events
- Temperature maintained at 37°C using a temperature-controlled environment (Tokai Hit)
- Cells imaged using TIRFM setup
- Images captured using Photometrics Prime 95B camera mounted on a Nikon Ti-2 microscope using Apo-TIRF 100×/1.49 oil-immersion or Apo-TIRF 60×/1.49 oil-immersion objective
- Image acquisition performed using NIS Elements Advanced Research Imaging software (version 4.60.00 [Build 1171] Patch 02)
- No positive or negative controls were explicitly mentioned in the provided information.
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