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Autophagy Detection Probe Blue

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RUO
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Autophagy_Detection_Probe_Blue_080123
Fluorescence image overlays of lung carcinoma cell line, A549, stained with the Autophagy Detection Probe Blue (cyan) and DRAQ5™ (magenta) (Cat. No. 424101). Cells grown in complete media (non-starved) show very weak fluorescence in autophagosomes (left). Cells which have undergone starvation in serum-free, glucose free media for 4 hours show fluorescence in autophagosomes (cyan) (right). Images were acquired using a 60X objective with DAPI and Cyanine5 filter cubes. Scale Bar = 20 µm
  • Autophagy_Detection_Probe_Blue_080123
    Fluorescence image overlays of lung carcinoma cell line, A549, stained with the Autophagy Detection Probe Blue (cyan) and DRAQ5™ (magenta) (Cat. No. 424101). Cells grown in complete media (non-starved) show very weak fluorescence in autophagosomes (left). Cells which have undergone starvation in serum-free, glucose free media for 4 hours show fluorescence in autophagosomes (cyan) (right). Images were acquired using a 60X objective with DAPI and Cyanine5 filter cubes. Scale Bar = 20 µm
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421906 1 vial 500 CHF
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Description

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 Blue 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
Technical Data Sheet (pdf)

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: Phosphate buffer saline, pH7.2 (PBS), phenol-free media, Hanks Balanced Salt solution (HBSS), or Cell Staining Buffer (Cat. No. 420201)

Imaging Guidelines:
Ex/Em = 330/520 nm 
Fluorescence microscope filter set: DAPI

Assay Protocol:

  1. Thaw the probe at room temperature before starting the experiment.
  2. Treat your cells with test compounds or conditions (e.g. starvation, Rapamycin, Chloroquine, Autophinib).
  3. 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.
  4. Remove media from cells and add the desired volume of autophagy staining solution. If cells are in suspension, spin down and resuspend.
  5. Optional: Include live-cell nuclear counterstain such as DRAQ5™ (Cat. No. 424101).
  6. Incubate cells at 37°C for 15-60 minutes.
  7. Remove autophagy staining solution. If cells are in suspension, spin down and remove solution.
  8. Gently wash cells twice with Cell Staining Buffer (Cat. No. 420201), PBS, phenol-free media, or HBSS.
  9. Immediately proceed to imaging.

Antigen Details

Distribution

Autophagosomes

Biology Area
Cell Biology, Cell Structure, Protein Trafficking and Clearance
Molecular Family
Autophagosome Markers
Antigen References
  1. Aman Y, et al. 2021. Nat Aging. 1:634–50.
  2. Dikic I, et al. 2018. Nat Rev Mol Cell Biol. 19:349–64.
  3. Khandia R, et al. 2019. Cells. 8:674.
Gene ID
NA
Go To Top Version: 1    Revision Date: 08.01.2023

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

BioLegend, the BioLegend logo, and all other trademarks are property of BioLegend, Inc. or their respective owners, and all rights are reserved.

 

8999 BioLegend Way, San Diego, CA 92121 www.biolegend.com
Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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