DALGreen - Autophagy DetectionD675 Product Manual

  Autophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles. In this process, an isolation membrane composed of a double membrane appears in cytosol, expands gradually, enfolds with the aggregated proteins and damaged organelles, and closes to form autophagosomes. The autophagosomes are fused with lysosomes to form autolysosomes, in which an acidic environment exists. The contents in autolysosomes are decomposed by digestive enzymes in lysosomes. Since this cellular function is said to be related to aging as well as neurodegenerative diseases such as Parkinson’s disease, a simple autophagy detection method is required.
  DALGreen is a small fluorescent molecule. Because it has unique properties which emits fluorescence under hydrophobic and acidic conditions, DALGreen can detect the autolysosomes. DALGreen is cell permeable, has no requirement of transfection method, and enables live cell imaging with fluorescence microscopy and quantitative assay by flow cytometry.

Fig. 1 The detection of autophagy with DALGreen

DALGreen - Autophagy Detection

20 nmol x 1

 

Store at -20^oC and protect from light.

• Dimethyl sulfoxide (DMSO)
• Culture medium
• Hanks’ HEPES buffer or serum-free medium
• Micropipettes

Preparation of 1 mmol/l DALGreen DMSO stock solution

Add 20 μl of DMSO to a tube of DALGreen (20 nmol) and dissolve it with pipetting.

• Store the reconstituted DMSO solution at -20^oC. The solution is stable at -20^oC for 1 month.

Preparation of DALGreen working solution

Dilute the 1 mmol/l DALGreen DMSO stock solution with culture medium to prepare 0.1-1.0 μmol/l DALGreen working solution.

• Please optimize the final concentration of DALGreen depeneding on the cell lines.

Autophagy detection

1. Prepare cells on dish for assay.
2. Discard the supernatant and wash the cells with culture medium.
3. Add an appropriate volume of DALGreen working solution and then incubate at 37^oC for 30 minutes.
4. Discard the supernatant and wash the cells with culture medium twice.
5. Add medium containing autophagy-inducing agent and incubate at 37^oC.
   • Please optimize the incubation time according to autophagy-inducing conditions.
6. Observe fluorescence with a fluorescence microscope or flow cytometer.

Observation on Confocal Fluorescence Microscopy

HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37^oC overnight in a 5% CO₂ incubator. The cells were washed with culture medium and then incubated at 37^oC for 30 minutes with 250 μl of 1 μmol/l DALGreen working solution. After the cells were washed with the culture medium twice, the culture medium or amino acid-free mediumthe (FUJIFILM Wako Pure Chemical, Ltd., Code: 048-33575) was added to the well. After 6 hours incubation, the cells were washed with Hanks’ HEPES buffer twice and then DALGreen was observed by confocal fluorescence microscopy.

Fig. 2 Confocal microscopic images of various autophagy induced conditions.
After the addition of DALGreen, the cells were incubated with the culture medium (A) or the amino acid-free medium (B).
Fluorescence images were obtained using confocal microscopy at an excitation wavelength of 488 nm and a 500-563 nm emission filter. Scale bar: 20 μm.

 

Analysis by Flow Cytometry

HeLa cells were seeded on 24 well plate and cultured at 37^oC overnight in a 5% CO₂ incubator. The cells were washed with culture medium and then incubated at 37^oC for 30 minutes with 1 μmol/l DALGreen working solution. After the cells were washed with the culture medium twice, the culture medium or amino acid-free medium was added to the well. After 20 hours incubation, the cells were washed with PBS, treated with trypsin and centrifuged. The pellets were suspended in Hanks’ HEPES buffer, and detected by flow cytometry.

Excitation and emission spectra of DALGreen

λex : 405 nm λem : 525 nm Ex：350 ～ 450 nm Em：500 ～ 560 nm