DALGreen - Autophagy Detection

∼ Features ∼

  1. Allow to Monitor Autophagy at Real Time
  2. Detect Autolysosome selectively in Live Cells
  3. Very simple procedure. Just add the working solution reagent to cultured cells


 


Content: 20 nmol x 1

Storage Condition :Store at -20 oC and protect from light

Shipping Condition :Ambient Temperature


Product Description
Simple Procedure
Procedure Comparison between DALGreen and Existing Reagents
Comparison to LC3
Comparison to MDC
Time-lapse imaging of autophagy
The Number of Usable Assay
References


Product Description

DALGreen is used to detect autophagy in live cells. Autophagy is an intracellular degradation system, where dysfunctional proteins and organelles are degraded. In this process, aggregated dysfunctional proteins are surrounded by the double membrane to form an autophagosome. DALGreen, which is a small hydrophobic molecule, passes the plasma membrane of live cells and is incorporated in the autophagosome. After a lysosome fuses with the autophagosome, the environment in the autolysosome become acidic. DALGreen fluoresce stronger as acidity increases. The quality of this dye enables live cell imaging with a fluorescence microscopy and quantitative assay by flow cytometry.


When an autophagosome membrane is formed, DALGreen is incorporated inside of the autophagosome membrane when the membrane is formed. The fluorescence of DALGreen is enhanced under acidic condition after the autophagosome is fused with the lysosome.



Simple Procedure

First, cells are plated for the assay. After discarding the supernatant and washing the cells with culture medium, DALGreen working solution is added. While incubating for 30 minutes, DALGreen passes the plasma membrane of live cells and is incorporated in the forming autophagosome (Fig.1). After the 30 minutes incubation, supernatant is removed and the cells are washed. Medium containing autophagy-inducing agent is added to the cells. After incubating, observation can be made via fluorescence microscope or flow cytometer. For more detailed procedure, please refer to the manual.




Procedure Comparison between DALGreen and Existing Reagents




Comparison to LC3


Method of autophagy induction
Control: incubated in the cell media for 6 hours
Starved: incubated in media without amino acid for 6 hours

Condition of DALGreen Imaging
Cell line: HeLa
Wavelength: Ex. 488 nm/ Em. 500-563 nm
Scale bar: 20 μm


Comparison to MDC

DALGreen can be used for live autophagy monitoring because the Working Solution is added prior to inducing autophagy unlike MDC.



Time-lapse imaging of autophagy


For more details, click application data


The Number of Usable Assay



Microscopy: 250 μl /assay (8 well chamber slide)
Flow cytometry: 500 μl (24 well plate)

[Condition]
・Final concentration of DALGreen working solution: 1.0 μmol/l
・The total volume prepared at 1.0 μmol/l DALGreen working solution: 20 ml

NOTE: The number of assay depends on the final concentration of DALGreen or volume of working solution.


References

1) H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, "Small fluorescent molecules for monitoring autophagic flux", FEBS Lett., 2018, 592, (4), 559–567.

2) T. Sakata, A. Saito and H. Sugimoto, "In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing", Scientific Reports., 2018, 8, 8282.

3) Y. Egawa, C. Saigo, Y. Kito, T. Moriki and T. Takeuchi , "Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma", PLoS One., 2018, 13, (6), e0198940.


Item # Description/Size Availability Qty Break Price Quantity
D675-10
1 set (20 nmol x 1)
1-2 business days 1 $280.00

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For Research Use Only Products