Cell Meter™ Autophagy Assay Kit *Blue Fluorescence*

Protocol summary

1. Prepare cells with your test compounds at the density of 1 - 2 × 10⁴ cells/well
2. Add Autophagy Blue™ working solution
3. Incubate at 37°C for 15 - 60 minutes
4. Wash the cells with Wash Buffer
5. Monitor the fluorescence increase at Ex/Em= 330/520 nm (Cutoff = 475 nm), fluorescence microscope with DAPI filter set

Important notes
Thaw all the components at room temperature before starting the experiment.

Add 20 μL of 500X Autophagy Blue™ (Component A) into 10 mL of Stain Buffer (Component B) and mix well to make Autophagy Blue™ working solution. Protect from light. Note: 20 μL of 500X Autophagy Blue™ (Component A) is enough for one 96-well plate.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Culture cells to a density optimal for autophagy induction according to your specific induction protocol (about 1 - 2 x 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. Remove medium.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Autophagy Blue™ working solution into each well.
   
   
4. Incubate the cells in a 37°C, 5% CO₂ incubator for 15 to 60 minutes. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
5. Wash the cells with Wash Buffer (Component C) for 3 - 4 times, then 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.
   
   
6. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 330/520 nm (Cutoff = 475 nm), a fluorescence microscope with DAPI filter set.