Autophagic Flux Assay Kit |
| Catalog No.GC26764 |
Autophagy is the process of autonomous degradation of cytoplasmic components such as proteins and organelles.
Products are for research use only. Not for human use. We do not sell to patients.
Sample solution is provided at 25 µL, 10mM.
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Autophagy is the process of autonomous degradation of cytoplasmic components such as proteins and organelles. Studies have found that it plays an important role in the study of aging, neurodegenerative diseases, etc. As research deepens, many researchers need to confirm the relationship between the autophagic process and the induction of autophagy or the inhibition of autophagic lysosomes, so the study of autophagic flux is crucial.
This kit contains autophagosome and autophagic lysosome detection dye (DAPRed), autophagosome detection dye (DALGreen) and lysosomal acidification inhibitor (bafilomycin A1), which can accurately detect the early autophagic process, autophagic lysosomes and the final digestion and degradation process. [1]
This kit contains (DAPRed - Autophagy Detection) for detecting autophagosomes and autophagic lysosomes, (DALGreen - Autophagy Detection) for detecting autophagic lysosomes and lysosomal acidification inhibitor Bafilomycin A1. By adding these reagents, you can experiment the entire process from autophagosome formation to autophagic lysosomes. [2][3]
References:
[1] H. Sakurai, et al., "Development of small fluorescent probes for the analysis of autophagy kinetics“, iScience, 2023, 26, 107218.
[2] X. Chen, et al, "Chaiqi decoction ameliorates vascular endothelial injury in metabolic syndrome by upregulating autophagy, Am J Transl Res., 2020, 12(9):4902-4922.
[3] C. Oh, et al, "S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia, J Neurosci., 2022, 42(14), 3011-3024.
Experimental Example 1: Observation of the inhibitory effect of Bafilomycin A1 on autophagic lysosome formation using confocal microscopy
Use DALGreen and DAPRed to label HeLa cells to evaluate the effect of lysosomal acidification inhibitor Bafilomycin A1 (Baf. A1) on autophagic flux. Fluorescence detection showed that compared with the starvation treatment group, the group treated with Baf.A1 inhibited the generation of autophagosomes and reduced the fluorescence intensity of DALGreen. On the contrary, there is no difference in the fluorescence generation of DAPRed. The experimental results showed that Baf. A1 can lead to the accumulation and stagnation of autophagosomes.
*The concentration, stimulation time, and timing of addition of Bafilomycin A1 may have an impact on the final imaging results. It is recommended to refer to the Baf.A1 processing section in the "Frequently Asked Questions Q&A" section of the webpage.
<Experimental conditions>
CTRL: negative group, STV: starvation induced autophagy group, Stv.+Raf. A1: autophagosome inhibition group
DALGreen fluorescence settings: 488 nm (Ex), 490-550 nm (Em)
DAPRed fluorescence settings: 561 nm (Ex), 565-700 nm (Em)
<Operation steps>
1. Inoculate HeLa cells with a density of 1.0 * 104/well into a μ - slide 8-well plate and culture overnight at 37 ℃ in a 5% CO2 incubator.
2. After washing twice with MEM (10% fetal bovine serum), add 200 μ l of DALGreen/DAPRed mixed working solution (DALGreen: 1 µ mol/l, DAPRed: 0.2 µ mol/l) and incubate at 37 ℃ for 30 minutes.
3. Discard the supernatant and wash twice with MEM (10% fetal bovine serum).
4. Sample preparation refers to the following conditions.
·Add 200 μ l of MEM (10% fetal bovine serum) to the well plate and incubate at 37 ℃ for 2 hours and 20 minutes. (Control group)
·Add 200 μ l of amino acid free medium to the orifice plate and culture at 37 ℃ for 2 hours and 20 minutes. (Hunger induced autophagy group)
·Add 200 μ l of amino acid free culture medium to the well plate, incubate at 37 ℃ for 2 hours, discard the supernatant, and add 200 μ l of lysosome acidification inhibitor bafilomycin A1 working solution. Incubate at 37 ℃ for 20 minutes. (Autophagy lysosome inhibition group)
5. Detect cells under a confocal laser microscope.
Experimental Example 2: Observation of the inhibitory effect of 3-MA on autophagosome formation using laser confocal microscopy
Use DALGreen and DAPRed to label HeLa cells to evaluate the effect of autophagy inhibitor 3-MA on autophagy flow. Compared with the starvation treatment group, the fluorescence intensity of DALGreen and DAPRed decreased in the treatment group with the addition of 3-MA, confirming that 3-MA inhibits the generation of autophagosomes.
*The stimulation time and added time points of 3-MA may have an impact on the final imaging results. It is recommended to refer to the 3-MA processing section in the "Frequently Asked Questions Q&A" section of the webpage.
<Experimental conditions>
CTRL: negative group, STV: starvation induced autophagy group, Stv.+3-MA: autophagosome inhibition group
DALGreen fluorescence settings: 488 nm (Ex), 490-550 nm (Em)
DAPRed fluorescence settings: 561 nm (Ex), 565-700 nm (Em)
<Operation steps>
1. Inoculate HeLa cells with a density of 1.0 * 104/well into a μ - slide 8-well plate and culture overnight at 37 ℃ in a 5% CO2 incubator.
2. After washing twice with MEM (10% fetal bovine serum), add 200 μ l of DALGreen/DAPRed mixed working solution (DALGreen: 1 µ mol/l, DAPRed: 0.2 µ mol/l) and incubate at 37 ℃ for 30 minutes.
3. Discard the supernatant and wash twice with MEM (10% fetal bovine serum).
4. Sample preparation refers to the following conditions.
·Add 200 μ l of MEM (10% fetal bovine serum) to the well plate and incubate at 37 ℃ for 2 hours and 20 minutes. (Control group)
·Add 200 μ l of amino acid free medium to the orifice plate and culture at 37 ℃ for 2 hours and 20 minutes. (Hunger induced autophagy group)
·Add 200 μ l of amino acid free culture medium to the well plate, incubate at 37 ℃ for 2 hours, discard the supernatant, and add 200 μ l of 3-MA (10 mmol/l, autophagy inhibitor) working solution prepared in amino acid free culture medium. Incubate at 37 ℃ for 20 minutes. (Autophagosome inhibition group)
5. Detect cells under a confocal laser microscope.
Experimental Example 3: Observation of the inhibitory effect of E64d/Pepstatin A on lysosomal proteases using laser confocal microscopy
Use DALGreen and DAPRed to label HeLa cells to evaluate the effect of lysosomal protease inhibitor E64d/Pepstatin A (Pep A) on autophagic flux. Fluorescence detection showed that compared with the starvation treatment group, the treatment group with E64d/Pep A showed an increase in the fluorescence intensity of DALGreen and DAPRed, confirming that E64d/Pep A can lead to an increase in autolysosomes.
<Experimental conditions>
CTRL: negative group, STV: starvation induced autophagy group, Stv.+E64d/Pep A: lysosomal protease inhibitor group
DALGreen fluorescence settings: 488 nm (Ex), 490-550 nm (Em)
DAPRed fluorescence settings: 561 nm (Ex), 565-700 nm (Em)
<Operation steps>
1. Inoculate HeLa cells with a density of 1.0 * 104/well into a μ - slide 8-well plate and culture overnight at 37 ℃ in a 5% CO2 incubator.
2. After washing twice with MEM (10% fetal bovine serum), add 200 μ l of DALGreen/DAPRed mixed working solution (DALGreen: 1 µ mol/l, DAPRed: 0.2 µ mol/l) and incubate at 37 ℃ for 30 minutes.
3. Discard the supernatant and wash twice with MEM (10% fetal bovine serum).
4. Sample preparation refers to the following conditions.
·Add 200 μ l of MEM (10% fetal bovine serum) to the well plate and incubate at 37 ℃ for 2 hours. (Control group)
·Add 200 μ l of amino acid free medium to the orifice plate and culture at 37 ℃ for 2 hours. (Hunger induced autophagy group)
·Add 200 μ l of E64d/Pep A (10 µ g/ml each, lysosomal protease inhibitor) working solution prepared in amino acid free medium to the well plate. (Lysosomal protease inhibitor group)
5. Detect cells under a confocal laser microscope.
| Applications |
● No need for transfection, just add reagents to easily detect autophagic flux
● The kit contains the lysosomal acidification inhibitors required for the experiment |
| Shipping | Ship with Room temperature. |
| Storage Conditions | -20°C, protected from light, stored in nitrogen |
| Usage | For research use only! Not for use on humans. |
Average Rating: 5 (Based on Reviews and 30 reference(s) in Google Scholar.)
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