Overview

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1.           Culture cells to a density optimal for autophagy induction according to your specific induction protocol (about 1-2 × 10⁴ cells/ well/96-well plate). At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. 

2.       Prepare Autophagy Super Blue™ working solution by diluting 20 μL of Autophagy Super Blue™ (Component A) to 10 mL of Stain Buffer (Component B).

Note: 20 μL of 500 X Autophagy Super Blue™ (Component A) is enough for one 96-well plate. Aliquot and store unused 500 X Autophagy Super Blue™ at < -20ºC. Protect from light and avoid repeated freeze-thaw cycles.

3.       Remove medium, add 100 μL of Autophagy Super Blue™ working solution (from Step 2) into each well, and incubate the cells in a 37ºC, 5% CO₂ incubator for 15 min-1 hour.

Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.

4.       Wash the cells with Wash Buffer (Component C) for 3-4 times, add 100 μL Wash Buffer (Component C)  to each well.

Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.

5.       Monitor the fluorescent intensity with a fluorescence microscope or flow cytometer at Ex/Em = 330/520 nm.

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