Can I use any of your Cell Meter™ Autophagy Assay Kits with a flow cytometer?
Posted June 12, 2019
If so, which one shows the best response? Can you provide a sample protocol?
Yes, you can use any of our three Cell Meter™ Autophagy Assay Kits (Cat# 23000, 23001, 23002) with a flow cytometer, fluorescence microscope or fluorescence microplate reader. From our experience Cat# 23002 showed the best response when used in flow cytometric analysis, then Cat# 23001 and finally Cat# 23000.
Below is a sample autophagy assay protocol using a flow cytometer:
- Incubate Jurkat cells in RPMI medium with 10% FBS (control) or in 1X HBSS buffer with 5% FBS (starved) for 16 hours at 37°C.
- Spin down both the control and starved cells, and then re-suspend in 1X HBSS buffer.
- Prepare dye working solution by diluting 20 μL of Autophagy Blue™, Autophagy Super Blue™, or Autophagy Green™ (depending on which kit you are using) with 10 mL of Stain Buffer.
- Stain cells with dye working solution at room temperature for 20-30 minutes.
- After staining, spin down cells and wash with 1X HBSS buffer.
- Analyze cells with flow cytometer.
- Autophagy Blue™ has fluorescence excitation and emission at Ex/Em = 335/520 nm
- Autophagy Super Blue™ has fluorescence excitation and emission at Ex/Em = 335/520 nm
- Autophagy Green™ has fluorescence excitation and emission at Ex/Em = 485/530 nm