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Cell Meter™ Autophagy Assay Kit *Blue Fluorescence*

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. Cell Meter™ Autophagy Kit employs Autophagy Blue™ 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.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells with your test compounds at the density of 1 - 2 × 104 cells/well
  2. Add Autophagy Blue™ working solution
  3. Incubate at 37°C for 15 - 60 minutes
  4. Wash the cells with Wash Buffer
  5. Monitor the fluorescence increase at Ex/Em= 330/520 nm (Cutoff = 475 nm), fluorescence microscope with DAPI filter set

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

PREPARATION OF WORKING SOLUTION

Add 20 μL of 500X Autophagy Blue™ (Component A) into 10 mL of Stain Buffer (Component B) and mix well to make Autophagy Blue™ working solution. Protect from light. Note: 20 μL of 500X Autophagy Blue™ (Component A) is enough for one 96-well plate.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Culture cells to a density optimal for autophagy induction according to your specific induction protocol (about 1 - 2 x 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 Blue™ 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 = 330/520 nm (Cutoff = 475 nm), a fluorescence microscope with DAPI filter set.

Citations

View all 15 citations: Citation Explorer
Modification of BCLX pre-mRNA splicing has antitumor efficacy alone or in combination with radiotherapy in human glioblastoma cells
Authors: Dou, Zhihui and Lei, Huiwen and Su, Wei and Zhang, Taotao and Chen, Xiaohua and Yu, Boyi and Zhen, Xiaogang and Si, Jing and Sun, Chao and Zhang, Hong and others,
Journal: Cell Death \& Disease (2024): 160
YAP promotes the healing of ischemic wounds by reducing ferroptosis in skin fibroblasts through inhibition of ferritinophagy
Authors: Cao, Guoqi and Yin, Siyuan and Ma, Jiaxu and Lu, Yongpan and Song, Ru and Wu, Zhenjie and Liu, Chunyan and Liu, Jian and Wu, Peng and Sun, Rui and others,
Journal: Heliyon (2024)
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
Cell cycle arrest is an important mechanism of action of compound Kushen injection in the prevention of colorectal cancer
Authors: Sun, Jie and Li, Mei and Lin, Tingru and Wang, Di and Chen, Jingyi and Zhang, Yu and Mu, Qing and Su, Huiting and Wu, Na and Liu, Aiyu and others,
Journal: Scientific reports (2022): 1--16
GDF-15 Deficiency Reduces Autophagic Activity in Human Macrophages In Vitro and Decreases p62-Accumulation in Atherosclerotic Lesions in Mice
Authors: Heduschke, Aline and Ackermann, Kathrin and Wilhelm, Beate and Mey, Lilli and Bonaterra, Gabriel Alejandro and Kinscherf, Ralf and Schwarz, Anja
Journal: Cells (2021): 2346

References

View all 28 references: Citation Explorer
beta-Elemene induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549 cells
Authors: Liu J, Hu XJ, Jin B, Qu XJ, Hou KZ, Liu YP.
Journal: J Pharm Pharmacol (2012): 146
Tgf-beta1 induces autophagy and promotes apoptosis in renal tubular epithelial cells
Authors: Xu Y, Yang S, Huang J, Ruan S, Zheng Z, Lin J.
Journal: Int J Mol Med. (2012)
High-Throughput Screening for AntiInfluenza A Virus Drugs and Study of the Mechanism of Procyanidin on Influenza A VirusInduced Autophagy
Authors: Dai J, Wang G, Li W, Zhang L, Yang J, Zhao X, Chen X, Xu Y, Li K.
Journal: J Biomol Screen. (2012)
An autophagy inhibitor enhances the inhibition of cell proliferation
Authors: Yao F, Wang G, Wei W, Tu Y, Tong H, Sun S.
Journal: Mol Med Report (2012): 84
Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells
Authors: Zhang YH, Wu YL, Tashiro S, Onodera S, Ikejima T.
Journal: Acta Pharmacol Sin (2011): 1266
Page updated on October 11, 2024

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Catalog Number23000
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Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationDAPI channel
EmissionDAPI channel
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation330 nm
Emission520 nm
Cutoff475 nm
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode

Components

Autophagy Blue&trade; labeled vesicles are induced by starvation in HeLa cells using Cell Meter&trade; Autophagy Assay Kit. HeLa cells were incubated in a regular DMEM medium as control (A) or in a serum-depleted medium as autophagy treatment (B) for 16 hours. Both control cells and starved cells were incubated with Autophagy Blue&trade; working solution for 30 minutes in a 37 &deg;C, 5%&nbsp; CO<sub>2&nbsp;</sub>incubator, and then washed four times with wash buffer. Cells were imaged immediately under a fluorescence microscope with a DAPI channel. Autophagy is indicated by bright blue dot staining of autophagic vacuoles (B).
Autophagy Blue&trade; labeled vesicles are induced by starvation in HeLa cells using Cell Meter&trade; Autophagy Assay Kit. HeLa cells were incubated in a regular DMEM medium as control (A) or in a serum-depleted medium as autophagy treatment (B) for 16 hours. Both control cells and starved cells were incubated with Autophagy Blue&trade; working solution for 30 minutes in a 37 &deg;C, 5%&nbsp; CO<sub>2&nbsp;</sub>incubator, and then washed four times with wash buffer. Cells were imaged immediately under a fluorescence microscope with a DAPI channel. Autophagy is indicated by bright blue dot staining of autophagic vacuoles (B).
Autophagy Blue&trade; labeled vesicles are induced by starvation in HeLa cells using Cell Meter&trade; Autophagy Assay Kit. HeLa cells were incubated in a regular DMEM medium as control (A) or in a serum-depleted medium as autophagy treatment (B) for 16 hours. Both control cells and starved cells were incubated with Autophagy Blue&trade; working solution for 30 minutes in a 37 &deg;C, 5%&nbsp; CO<sub>2&nbsp;</sub>incubator, and then washed four times with wash buffer. Cells were imaged immediately under a fluorescence microscope with a DAPI channel. Autophagy is indicated by bright blue dot staining of autophagic vacuoles (B).