Autophagy Inhibitor, 3-MA

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	- 200 µM concentration of 3-MA can be used to treat the cells

Protocol tips
- 200 µM concentration of 3-MA can be used to treat the cells

Publication protocol

The procedure used to transfect and evaluate adenoviral mRFP-GFP-LC3 has been described previously [29]. Briefly, the day before transfection, good quality BEAS-2B cells were seeded into 24-well plates and cultured overnight. The next day, and at a cell density of between 50–70%, viral infection was performed. The adenoviral mRFP-GFP-LC3 was removed from the -80°C freezer in advance and thawed on ice. Transfection steps were performed in strict accordance with the manufacturer’s instructions. BEAS-2B cells were transfected with adenoviral mRFP-GFP-LC3 at a multiplicity of infection (MOI) value of 50. The medium was refreshed 6 h later and the cells were cultured for a further 24 h. Transfected BEAS-2B cells were treated with propranolol (10 μM), ICI118551 (1 μM), rapamycin (50 nM), and 3-MA (200 μM) for 1 h prior to CSC intervention (300 mg/L). Cells were then left to incubate for an additional 24 h. Cells were then fixed with 4% paraformaldehyde and viewed under a laser scanning confocal microscope (Olympus, Tokyo, Japan). The yellow spots which appeared after merge indicated autophagosomes, the red spots indicated autolysosomes.The intensity of autophagic change could be determined by counting the different colored fluorescent dots on confocal microscopy. We use an artificial counting method, and at least 50 cells were counted in each experiment.

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