AGS cells treated at different pH (6.5 and 7.4) were seeded on coverslips and grown to reach 50% confluence. They were then infected with mRFP-GFP-LC3 adenoviruses (HanBio, shanghai) according to the manufacturer’s guidelines. The medium was replaced with different pH medium after 24 h. Cells were then fixed with 4% paraformaldehyde. The fixed cells were treated with 5% BSA for 30 min and stained with DAPI (Solarbio, Tongzhou Dist. Beijing) for 6 min. The coverslips were then observed by laser confocal microscopy (×189 magnification), as described previously (25 (link)). We counted the number of mRFP-GFP-LC3 puncta in three independent visual fields and analyzed the results using Image J software.
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Autophagy Visualization in AGS Cells
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Corresponding Organization : Affiliated Hospital of Qingdao University
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Variable analysis
independent variables
- PH of the medium (6.5 and 7.4)
- Number of mRFP-GFP-LC3 puncta in three independent visual fields
- Cell type (AGS cells)
- Cell seeding density (50% confluence)
- Infection with mRFP-GFP-LC3 adenoviruses
- Fixation with 4% paraformaldehyde
- Blocking with 5% BSA
- Staining with DAPI
- Microscopy method (laser confocal microscopy, ×189 magnification)
- No positive or negative controls were explicitly mentioned in the protocol.
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