- Regulatory Status
- RUO
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Fluorescence image overlays of lung carcinoma cell line, A549, stained with the Autophagy Detection Probe Green and Hoechst 3342 (blue). Cells grown in complete media (non-starved) show very weak green fluorescence in autophagosomes (left). Cells which have undergone starvation in serum-free and glucose-free media for 4 hours show green fluorescence in autophagosomes (right). Images were acquired using a 60X objective with FITC and DAPI filter cubes. Scale bar = 20 µm
| Cat # | Size | Price | Quantity Check Availability | ||
|---|---|---|---|---|---|
| 421904 | 1 vial | $500.00 | |||
Autophagy is a highly conserved degradation process that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. In the absence of cellular stress, autophagy serves as a house-keeping function, removing damaged organelles and cellular components. Insufficient autophagy leads to accumulation of damaged cells and aging. When activated during cellular stress or starvation, the breakdown of cellular components promotes cellular survival by maintaining cellular energy levels. Autophagy Detection Probe Green employs a specific autophagosome marker to analyze the activity of autophagy. The assay is optimized for direct detection of autophagy in live cells.
Product Details
- Verified Reactivity
- Human
- Reported Reactivity
- Mouse
- Formulation
- 1 vial of Autophagy Detection Probe (50 µL)
- Preparation
- Please refer to detailed protocol in application notes.
- Storage & Handling
- -20°C
- Application
-
Live cell imaging - Quality tested
- Recommended Usage
-
Please refer to detailed protocol in application notes.
- Application Notes
-
This probe employs a specific autophagosome marker for detection of autophagy in live cells (adherent or suspension). Using the recommended volume of 100 μL per well in a 96-well plate, this product supplies enough reagents for 200 tests.
Component:
50 μL of 100X Autophagy Probe (-20°C, protected from light, avoid freeze/thaw cycles)
Required Materials Not Included:
Stain Buffer: PBS, phenol-free media, HBSS, or Cell Staining Buffer (Cat. No. 420201)
Imaging Guidelines:
Ex/Em = 485/530 nm
Fluorescence microscope filter set: FITC
Assay Protocol:- Thaw the probe at room temperature before starting the experiment.
- Treat your cells with test compounds or conditions (e.g. starvation, Rapamycin, Chloroquine, Autophinib).
-
Prepare the autophagy staining solution by diluting the autophagy probe 1:500 in Cell Staining Buffer (Cat. No. 420201), PBS, phenol-free media, or HBSS and mix well.Note: We recommend 100 μL per well in a 96-well plate.
- Remove media from cells and add the desired volume of autophagy staining solution. If cells are in suspension, spin down and resuspend.
- Optional: Include live-cell nuclear counterstain such as DRAQ5™ (Cat. No. 424101).
- Incubate cells at 37°C for 15-60 minutes.
- Remove autophagy staining solution. If cells are in suspension, spin down and remove solution.
- Gently wash cells twice with Cell Staining Buffer (Cat. No. 420201), PBS, phenol-free media, or HBSS.
- Immediately proceed to imaging.
Antigen Details
- Distribution
-
Autophagosomes
- Biology Area
- Cell Biology, Cell Structure, Protein Trafficking and Clearance
- Molecular Family
- Autophagosome Markers
- Antigen References
-
- Aman Y, et al. 2021. Nat Aging. 1:634–50.
- Dikic I, et al. 2018. Nat Rev Mol Cell Biol. 19:349–64.
- Khandia R, et al. 2019. Cells. 8:674.
- Gene ID
- NA