High Correlation with LC3

Result:
Almost all DAPGreen signals were colocalized with LC3.

Imaging Condition:
DAPGreen:Ex. 488 nm/Em. 500-563nm
Scale bar: 10 μm

The Condition of Autophagy Induction:
After adding DAPGreen to the RFP-LC3 expressed Hela cells, cells were treated with rapamycin to induce autophagy. Fluorescent imaging was conducted by confocal microscopy after 4 hrs. from autophagy induction.

Quantitative Analysis by Flow Cytometer

Result:
After 3 hrs. of incubation under starved condition, strong fluorescence was detected.

Detection:
Wavelengths: Ex. 488 nm / Em. 500-560 nm

The Condition of Autophagy Induction:
After staining with DAPGreen, HeLa cells were incubated for 0, 3, 6 hrs. with amino acid-free medium and detected by a flow cytometer.

Quantitative Analysis by Microplate Reader

Result:
After 2hrs. of incubation under starved condition fluorescence was observed. It was ca. 3,5 times stronger than “Control”.

Detection:
Wavelengths: Ex. 450nm/ Em. 535nm

The Condition of Autophagy Induction:
After staining with DAPGreen, HeLa cells were incubated for 0, 2, 4, 6 hrs. with amino acid-free medium and detected by a microplate reader.

DAPGreen Excitation/Emission

Usage examples from Publications

Microscopy: 250 μl /assay (8 well chamber slide)
Flow cytometry: 2,000 μl (6 well plate)
Plate reader: 100 μl/assay (96 well plate)

[Condition] ・Final concentration of DAPGreen working solution: 0.1 μmol/l
・The total volume prepared at 0.1 μmol/l DAPGreen working solution: 50 ml

 

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

Related Product Information

Detection is possible with a fluorescent microscope, a flow cytometer and a microplate reader using DAPGreen and DAPRed [#D677]. DALGreen [#D675] detects autolysosome. After a lysosome fuses with the autophagosome, the environment in the autolysosome become acidic. DALGreen fluoresce stronger as acidity increases. DALGreen can be applied in two methods (a fluorescent microscope and a flow cytometer). Please select your most suitable method depending on your equipment.

Quantification by fluorescence microscopy

HeLa cells in a 96-well plate were stained with DAPGreen. Then, autophagy was induced through serum starvation before being detected via fluorescence microscopy.
The mean fluorescent intensity (MFI) per field of view was then quantified, using the Nikon software NIS-Elements. The results indicated that the MFI was increased in the serum-free HeLa cells.

＜Detection conditions＞

Ex = 488 nm, Em = 500-550 nm

Microscope Objective Lens:  CFI Plan Apochromat VC 20x

Operation: Resonant Scanning

Lateral resolution (x-y): 512 x 512

 

＜Equipment＞

Confocal Microscope: Nikon A1R

Software: NIS-Elements

 

＜Procedure＞

1. Prepare HeLa cells for the assay in 96-well microplates and incubate at 37oC for overnight.

2. Wash once with culture medium (with serum) after removing the supernatant.

3. Add DAPGreen working solution and incubate at 37oC for 30 minutes.

4. Wash twice with culture medium (with serum) after removal of supernatant.

5. Add medium (with or without serum) to the wells where autophagy is to be induced and incubate at 37oC for 6 hours.

6．Fix HeLa cells with 4% PFA and observe under a confocal microscope.

 

Furthermore, the cells were observed in more detail by switching to a 100X objective. The number of DAPGreen fluorescent puncta per cell were then counted, and an average of 27 puncta were detected in HeLa cells cultured in serum-free medium, while only the 1.5 fluorescent puncta per cell were detected with serum. This was a very significant increase compared to cells in serum-containing medium.

＜Detection conditions＞

Ex = 488 nm, Em = 500-550 nm

Microscope Objective Lens: CFI Apochromat TIRF 100xC Oil

Operation:　Galvano Scanning

Lateral resolution (x-y): 512 x 512

 

＜Equipment＞

Confocal Microscope: Nikon A1R

Software: NIS-Elements

※This data was provided by the user.