Cell Meter™ Autophagy Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells with your test compounds
2. Add Autophagy Green™ working solution
3. Incubate at 37°C for 15 - 60 minutes
4. Wash cells with Wash Buffer
5. Monitor the fluorescence increase at Ex/Em= 485/530 nm (Cutoff = 515 nm), fluorescence microscope with FITC filter set or flow cytometer with 530/30 nm filter (FITC channel)

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

Add 20 μL of Autophagy Green™ (Component A) to 10 mL of Stain Buffer (Component B) and mix well to make Autophagy Green™ working solution. Protect from light. Note: 20 μL of 500X Autophagy Green™ (Component A) is enough for one 96-well plate. Note: Aliquot and store unused 500X Autophagy Green™ at < -20ºC. Protect from light and avoid repeated freeze-thaw cycles.

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

1. Culture cells to a density optimum 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. Remove medium.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Autophagy Green™ 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 = 485/530 nm (Cutoff = 515 nm), a fluorescence microscope with FITC filter set or a flow cytometer with 530/30 nm filter (FITC channel).