Cells seeded on glass coverslips, glass-bottom plates (Greiner Bio-One, Monroe, NC), or plastic-bottom plates (Ibidi, Fitchburg, WI) were fixed for 30 min in phosphate-buffered saline (PBS) containing 4% paraformaldehyde, washed in PBS, and then permeabilized with PBS containing 0.05% saponin and 5% FBS or with PBS containing 0.1% Triton X-100 and 5% FBS. Cells were stained as indicated. Images were collected using a Nikon Eclipse Ti2 inverted epifluorescence microscope fitted with a Nikon DS-Qi2 camera (Nikon Instruments Inc.) and a Lumencor Sola Pad excitation source (Lumencor, Beaverton, OR) or using a LSM710 confocal laser scanning microscope (Carl Zeiss Micro Imaging, Thornwood, NY). The ImageJ selection tool was used to measure CCV size and quantity. Automated image segmentation and analysis with CellProfiler were used to quantitate signal intensity and cell dimensions as previously described (41 (link)).
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Fluorescence Imaging of Cultured Cells
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Cell
Confocal laser scanning microscope
Microscope
Paraformaldehyde
Phosphate
Saline
Saponin
Triton x 100
Corresponding Organization :
Other organizations : National Institutes of Health, National Institute of Allergy and Infectious Diseases
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Variable analysis
independent variables
- Cell culture substrate (glass coverslips, glass-bottom plates, or plastic-bottom plates)
- CCV size
- CCV quantity
- Signal intensity
- Cell dimensions
- Phosphate-buffered saline (PBS)
- Paraformaldehyde concentration (4%)
- Saponin concentration (0.05%) or Triton X-100 concentration (0.1%)
- Fetal bovine serum (FBS) concentration (5%)
- No positive or negative controls were explicitly mentioned.
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