Cell Meter™ Autophagy Assay Kit Green Fluorescence

Autophagy Green™ labeled vesicles were induced by starvation in HeLa cells. HeLa cells were incubated in a regular DMEM medium (Left: Control) or in 1X HBSS buffer with 5% serum (Right: Autophagy Treatment) for 16 hours. Both control and starved cells were incubated with Autophagy Green™ working solution for 20 minutes in a 37 °C, 5% CO2 incubator, and then washed 3 times with wash buffer. Cells were imaged immediately under a fluorescence microscope with a FITC channel (green). Cell nuclei were stained with Hoechst 33342 (blue).

HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green™ (Cat No. 23002) working solution for 20 minutes in a 37°C, 5% CO<sub>2</sub> incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite™ Orange (Cat No. 22657).

HG-induced autophagy related gene expression and autophagosome formation. BMSCs in LG or HG were treated with MDC and examined under fluorescent microscopy. RA and 3MA denote rapamycin (5 μM) and 3-methlyadenine (10 μM), respectively. *Autophagosomes were detected with an autofluorescent compound monodansylcadaverine (MDC), using a Cell Meter Autophagy kit (AAT Bioquest, Inc.). Source: Graph from<strong> High Glucose Induces Bone Marrow-Derived Mesenchymal Stem Cell Senescence by Upregulating Autophagy</strong> by Tzu-Ching Chang, et al., <em>PLOS ONE</em>, May 2015.

Antioxidants abrogate HG-induced autophagy. Analysis of autophagy by MDC staining. *Autophagosomes were detected with an autofluorescent compound monodansylcadaverine (MDC), using a Cell Meter Autophagy kit (AAT Bioquest, Inc.). Source: Graph from <strong>High Glucose Induces Bone Marrow-Derived Mesenchymal Stem Cell Senescence by Upregulating Autophagy</strong> by Tzu-Ching Chang, et al., <em>PLOS ONE</em>, May 2015.

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Alternative formats

Cell Meter™ Autophagy Assay Kit *Blue Fluorescence*

Overview SDS Protocol

This Cell Meter™ Autophagy Kit employs Autophagy Green™ as a specific autophagosome marker to analyze the activity of autophagy. The assay is optimized for direct detection of autophagy in both detached and attached cells. The kit provides all the essential components for the assay protocol. Cell Meter™ Autophagy Kit is suitable for fluorescence microscope, fluorescence microplate reader and flow cytometer. Autophagy Green™ has a large Stokes shift. Autophagy is an evolutionarily conserved degradation process that targets long-lived proteins, organelles, and other cytoplasmic components for degradation via the lysosomal pathway. The autophagy pathway is complementary to the action of the ubiquitin-proteasome pathway which typically degrades short-lived proteins. Activation of the autophagy pathway is required for multiple cellular roles, including survival during starvation, the clearance of intracellular components, development, and immunity. In the absence of stress, autophagy serves a house-keeping function, removing damaged organelles and cellular components preventing cytotoxic effects. Decreases and defects in autophagy have been implicated in multiple diseases, for example Huntingtons, Alzheimers, and Parkinsons. In terms of cancer development, autophagy seems to play multiple roles. Decreased or absent expression of certain autophagy proteins, such as Beclin-1 and Bif-1, increases tumor susceptibility in mice while the overexpression of these proteins can repress cancer cell growth. However, autophagy is critical for the survival of cancer cells within the nutrient poor and hypoxic core of solid tumors.

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

Excitation	FITC filter
Emission	FITC filter
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	485 nm
Emission	530 nm
Cutoff	515 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with your test compounds
Add Autophagy Green™ working solution
Incubate at 37°C for 15 - 60 minutes
Wash cells with Wash Buffer
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.

PREPARATION OF WORKING SOLUTION

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

SAMPLE EXPERIMENTAL PROTOCOL

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.
Remove medium.
Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Autophagy Green™ working solution into each well.
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.
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.
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).

Images

Figure 1. Autophagy Green™ labeled vesicles were induced by starvation in HeLa cells. HeLa cells were incubated in a regular DMEM medium (Left: Control) or in 1X HBSS buffer with 5% serum (Right: Autophagy Treatment) for 16 hours. Both control and starved cells were incubated with Autophagy Green™ working solution for 20 minutes in a 37 °C, 5% CO2 incubator, and then washed 3 times with wash buffer. Cells were imaged immediately under a fluorescence microscope with a FITC channel (green). Cell nuclei were stained with Hoechst 33342 (blue).

Figure 2. HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green™ (Cat No. 23002) working solution for 20 minutes in a 37°C, 5% CO₂ incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite™ Orange (Cat No. 22657).

Figure 3. HG-induced autophagy related gene expression and autophagosome formation. BMSCs in LG or HG were treated with MDC and examined under fluorescent microscopy. RA and 3MA denote rapamycin (5 μM) and 3-methlyadenine (10 μM), respectively. *Autophagosomes were detected with an autofluorescent compound monodansylcadaverine (MDC), using a Cell Meter Autophagy kit (AAT Bioquest, Inc.). Source: Graph from High Glucose Induces Bone Marrow-Derived Mesenchymal Stem Cell Senescence by Upregulating Autophagy by Tzu-Ching Chang, et al., PLOS ONE, May 2015.

Figure 4. Antioxidants abrogate HG-induced autophagy. Analysis of autophagy by MDC staining. *Autophagosomes were detected with an autofluorescent compound monodansylcadaverine (MDC), using a Cell Meter Autophagy kit (AAT Bioquest, Inc.). Source: Graph from High Glucose Induces Bone Marrow-Derived Mesenchymal Stem Cell Senescence by Upregulating Autophagy by Tzu-Ching Chang, et al., PLOS ONE, May 2015.

Citations

View all 9 citations: Citation Explorer

Mitochondrial-Targeted Antioxidant MitoQ-Mediated Autophagy: A Novel Strategy for Precise Radiation Protection
Authors: Bao, Xingting and Liu, Xiongxiong and Wu, Qingfeng and Ye, Fei and Shi, Zheng and Xu, Dan and Zhang, Jinhua and Dou, Zhihui and Huang, Guomin and Zhang, Hong and others,
Journal: Antioxidants (2023): 453

Sarcopenia-derived exosomal micro-RNA 16-5p disturbs cardio-repair via a pro-apoptotic mechanism in myocardial infarction in mice
Authors: Hayasaka, Taiki and Takehara, Naofumi and Aonuma, Tatsuya and Kano, Kohei and Horiuchi, Kiwamu and Nakagawa, Naoki and Tanaka, Hiroki and Kawabe, Jun-ichi and Hasebe, Naoyuki
Journal: Scientific Reports (2021): 1--14

Effects of miR-34b/miR-892a upregulation and inhibition of ABCB1/ABCB4 on melatonin-induced apoptosis in VCR-resistant oral cancer cells
Authors: Hsieh, Ming-Ju and Lin, Chiao-Wen and Su, Shih-Chi and Reiter, Russel J and Chen, Andy Wei-Ge and Chen, Mu-Kuan and Yang, Shun-Fa
Journal: Molecular Therapy-Nucleic Acids (2020): 877--889

Effect and underlying mechanisms of airborne particulate matter 2.5 (PM2. 5) on cultured human corneal epithelial cells
Authors: Kashiwagi, Kenji and Iizuka, Yoko
Journal: Scientific reports (2020): 1--10

Effect and underlying mechanisms of airborne particulate matter 2.5 on cultured human corneal epithelial cells
Authors: Kashiwagi, Kenji and Iizuka, Yoko
Journal: Scientific reports (2020): 1--10

Coronarin D induces apoptotic cell death through the JNK pathway in human hepatocellular carcinoma
Authors: Lin, Hui-Wen and Hsieh, Ming-Ju and Yeh, Chao-Bin and Hsueh, Kuan-Chun and Hsieh, Yi-Hsien and Yang, Shun-Fa
Journal: Environmental toxicology (2018)

Methods for Measuring Autophagy Levels in Disease
Authors: Phadwal, Kanchan and Kurian, Dominic
Journal: (2017): 195--211

High glucose induces bone marrow-derived mesenchymal stem cell senescence by upregulating autophagy
Authors: Chang, Tzu-Ching and Hsu, Min-Fen and Wu, Kenneth K
Journal: PloS one (2015): e0126537

Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells
Authors: Tsai, Jen-Pi and Lee, Chien-Hsing and Ying, Tsung-Ho and Lin, Chu-Liang and Lin, Chia-Liang and Hsueh, Jung-Tsung and Hsieh, Yi-Hsien
Journal: Oncotarget (2015): 28851

References

View all 28 references: Citation Explorer

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Tgf-beta1 induces autophagy and promotes apoptosis in renal tubular epithelial cells
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High-Throughput Screening for AntiInfluenza A Virus Drugs and Study of the Mechanism of Procyanidin on Influenza A VirusInduced Autophagy
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An autophagy inhibitor enhances the inhibition of cell proliferation
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Inhibition of induced autophagy increases apoptosis of Nara-H cells
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Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells
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Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes
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Quantification of nonselective bulk autophagy in S. cerevisiae using Pgk1-GFP
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Concomitant inhibition of AKT and autophagy is required for efficient cisplatin-induced apoptosis of metastatic skin carcinoma
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