Autophagy Assay Kit (ab139484)
Key features and details
- Assay type: Cell-based
- Detection method: Fluorescent
- Platform: Microplate reader, Fluor. microscope, Flow cyt.
- Assay time: 30 min
- Sample type: Adherent cells, Suspension cells
製品の概要
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製品名
Autophagy Assay Kit
autophagy キット 製品一覧 -
検出方法
Fluorescent -
サンプルの種類
Adherent cells, Suspension cells -
アッセイタイプ
Cell-based -
全工程の試験時間
0h 30m -
製品の概要
Autophagy Assay Kit ab139484 measures autophagic vacuoles and monitors autophagic flux in live cells using a dye that selectively labels autophagic vacuoles. It can be analyzed using flow cytometry, fluorescent microscopy, or using a microplate reader. The dye has spectral characteristics similar to FITC.
The dye used in the autophagy assay protocol has been optimized by screening dyes for both minimal staining of lysosomes, and bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). The dye is a cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a similar manner to drugs that induce phospholipidosis.
This autophagy assay offers a rapid and quantitative approach to monitoring autophagy in live cells without the need for cell transfection.
Autophagy inducer rapamycin is included in the kit as a positive control. Please note that the optimal final concentration is cell-dependent and should be determined experimentally for each cell line being tested. The agent has been validated in HeLa, HepG2 and Jurkat cells.
Chloroquine is included in the kit to use as an inhibitor control. Chloroquine may be used in combination with rapamycin or starvation in monitoring autophagic flux. Depending on the applications and specific cell lines, it is recommended that treatment with the agent will be performed using 10-120 μM final concentration in order to observe changes in autophagic flux.
Please refer to the protocol book for more recommendations.
Autophagy assay protocol summary:
- remove growth medium from cells
- add staining mix and incubate for 30 min
- wash cells
- analyze with fluorescence microscopy, flow cytometry, or fluorescent microplate reader -
特記事項
The reagents provided in this kit are sufficient for 200 flow cytometry tests, 250 fluorescence microscopy test or 3 x 96-well microplate tests.
This product was previously called Autophagy Detection Kit.
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試験プラットフォーム
Microplate reader, Fluor. microscope, Flow cyt.
製品の特性
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保存方法
Store at -80°C. Please refer to protocols. -
内容 1 kit 10X Assay Buffer 1 x 30ml Autophagy Inducer 1 x 25nmole Chloroquine 1 x 7.5µmole Green Detection Reagent 1 x 50µl Hoechst 33342 Nuclear Stain 1 x 50µl -
研究分野
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別名
- autophagocitosis
画像
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Functional Assay: ab139484 Autophagy Detection Kit
Fluorescent microscopy analysis showing nucleus (blue nuclear stain; DAPI filter) and autophagic vesicules (green, FITC filter) in control HepG2 cells or cells treated with 1 uM Rapamycin (ab120224) for 24 hours.
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Functional Assay: ab139484 Autophagy Detection KitFluorescent microscopy analysis showing nucleus (blue nuclear stain; DAPI filter) and autophagic vesicules (green, FITC filter) in control HepG2 cells or cells starved in serum free medium for 5 hours to induce the formation of autophagic vesicles. HepG2 cells were also treated with 0.1 mM chloroquine for 24 hours or starved and chloroquine treated for 5 hours (in earlier stages of autophagy, chloroquine induces autophagosome formation).
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Flow cytometry-based profiling of ab139484Jurkat cells (acute T-Cell leukemia), uninduced or treated overnight with 0.5 µM Rapamycin (a typical autophagy inducer) were loaded with Green Detection Reagent, then washed and analyzed by flow cytometry. Results are presented as histogram overlay. Control cells (blue solid line) were stained as well but mostly display low fluorescence. In the samples treated with 500 nM Rapamycin for 18 hours (black solid line), Green dye signal increases about 2-fold, indicating that Rapamycin induced autophagy in Jurkat cells.
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (79)
ab139484 は 79 報の論文で使用されています。
- Kowluru RA et al. Impaired Removal of the Damaged Mitochondria in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy. Mol Neurobiol 61:188-199 (2024). PubMed: 37596436
- Park JH et al. Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy. Diabetes Metab J 48:215-230 (2024). PubMed: 37750184
- Chew HY et al. Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin. Sci Rep 14:4112 (2024). PubMed: 38374190
- Balasubramaniam M et al. Rescue of ApoE4-related lysosomal autophagic failure in Alzheimer's disease by targeted small molecules. Commun Biol 7:60 (2024). PubMed: 38191671
- Rajan PK et al. Normalization of the ATP1A1 Signalosome Rescinds Epigenetic Modifications and Induces Cell Autophagy in Hepatocellular Carcinoma. Cells 12:N/A (2023). PubMed: 37830582