You can appropriately extend the incubation time of Mtphagy Dye or Lyso Dye.
Example: Incubation time: 30 min → 60 min
If the staining fluorescence intensity is still not strong, please increase the concentration of the working solution.
Example: Mtphagy Dye concentration: 0.1 μM → 0.2～0.5 μM; Lyso Dye concentration: 1 μM → 2～5 μM
Select the appropriate filter for Mtphagy Dye.
Since the Stokes Shift of Mtphagy Dye is relatively large, the fluorescence wavelength of the filters on general instruments is not near the peak. If the sensitivity is insufficient, please use long-wavelength fluorescence filters.
Excitation: 550 nm±25 nm, Emission: 605±35 nm filters.
The medium used to induce autophagy can contain serum, but the working solution and staining should not contain serum. For the final detection, there are two scenarios:
Epifluorescence Microscope: The medium can contain serum, but it should not contain phenol red as it will increase the background.
Laser Confocal Microscope: No influence.
The fluorescence images of HeLa cells stained with Mtphagy Dye on our instruction of mitophagy detection kit manual were taken under a fluorescence microscope 24 hours after the addition of Mtphagy Dye to the cell culture. Experimental results show that the fluorescence intensity is strongest about 12 hours after adding Mtphagy Dye. Therefore, you can detect between 12-24 hours after adding Mtphagy Dye according to the actual situation.
Lyso Dye has a relatively short fluorescence quenching time, so it cannot be added together with Mtphagy Dye. It needs to be added before detection and incubated for 30-60 minutes for detection.
Because the dye first accumulates in normal mitochondria and stains autophagic lysosomes after apoptosis induction, experimental results show that the fluorescence intensity of fixed cells is relatively low, leading to poor detection results. Therefore, it is not recommended to use for staining fixed cells, and it cannot be used for staining dead cells.
The dye has decomposed due to moisture absorption and cannot enter the cells.
The staining time is too short or the dye concentration is too low.
No autophagy has occurred.
The detection wavelength is incorrect.
The medium contains serum, which interferes with the staining.
