Mitophagy detection kit

A Mitophagy Detection Kit (Dojindo Molecular Technologies) was used to detect mitophagy [22 (link)]. The chondrocytes were washed twice with DMEM and incubated with 100 nM Mtphagy Dye diluted in DMEM for 30 min at 37 °C. After incubation, cells were washed twice with DMEM and continued to be incubated for another 1 h in the previous culture conditions. After mitochondrial staining, the dye was immobilized and fluorescence intensity varied according to pH value. In mitochondrial-lysosome fusion, Mtphagy Dye displayed higher fluorescence intensity, indicating mitophagy. The level of mitophagy was defined by the area of Mtphagy Dye per cell.

The Mitophagy Detection Kit (Dojindo) was used to detect mitophagy. Briefly, HL60 and KG1a (5 × 10⁵ cells) were preincubated with Mitophagy Dye (30 minutes, 37°C), and then washed with Hank's Balanced Salt Solution (HBSS). The cells were then incubated for 6 hours with medium containing vehicle (DMSO), 5.0 μmol/L (HL60) or 10 μmol/L (KG1a) OR21, or 0.5 μmol/L (HL60) or 5.0 μmol/L (KG1a) Ven, or OR21 plus Ven (5.0 μmol/L+ 0.5 μmol/L or 10 μmol/L + 5.0 μmol/L). Next, 1 μmol/L of Lyso Dye was added for 30 minutes. After washing with HBSS, mitophagy was detected by flow cytometry or observed under a confocal microscope (LSM880; ZEISS). MFI was analyzed using FlowJo software.

Mitochondrial membrane potential was measured with JC-1 dye (ThermoFisher Scientific, Waltham, MA, USA) as described in [11 (link)]. Polarized mitochondria that accumulated more JC-1 dye were red. The reduction in the polarization was reflected by the increase in green fluorescence. The ratio of red to green fluorescence reflected the degree of the mitochondrial membrane polarization. The measurements were taken from at least 250 cells. For a positive control, the mitochondrial membrane was depolarized with 5 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling reagent.
The intensity of mitophagy in HL-1 cells with partially silenced FBP2 expression or treated with the 5 µM FBP2 inhibitor was assayed using the Mitophagy Detection Kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction. Briefly, cells were stained with Mtphagy Dye. The dye was immobilized in intact mitochondria and exhibited a weak fluorescence. When damaged mitochondria fused to lysosomes their fluorescence emission markedly increased. In a “positive control”, cells were treated with the mitophagy-stimulating concentration of CCCP (10 µm). Cells were observed in confocal microscopy and the intensity of fluorescence was measured as described above (“Confocal microscopy”). The measurements were taken from at least 400 cells.

The production of superoxides by the mitochondria was determined in live GMCs using the MitoSOX Red mitochondrial superoxide indicator Kit (Thermo Scientific). Live GMCs were loaded with 5 µM of the MitoSOX reagent and Hoechst 33342 nuclear stain for 10 min at 37 °C. Following 3 washes, the cells were immediately visualized, and random fluorescence images were documented using a Zeiss LSM 710 laser-scanning confocal microscope.
Mitophagy was documented in sparsely seeded GMCs using a mitophagy detection kit (Dojindo Molecular Technologies Inc., Rockville, MD, USA) following the manufacturer’s instructions. Briefly, the cells were washed with PBS and loaded with 100 nM of Mtphagy dye (mitophagy staining) for 30 min at 37 °C. The cells were then washed and treated with DMSO (control) or DOX for 18 h. The culture medium was removed, and cells were incubated in the dark with 1 µM Lyso dye (lysosome staining) at 37 °C for 30 min. The cells were washed with PBS, after which the co-localization between Mtphagy (Ex. 561 nM/Em. 650 nM) and Lyso (Ex. 488 nM/Em. 502–554 nM) dyes were documented with a Zeiss LSM 710 laser-scanning confocal microscope.

Cardiomyocyte mitophagy was detected by Mitophagy Detection Kit (Dojindo). Cardiomyocytes were cultured in 37 °C with 100 nmol/L Mtphagy dyes for 30 min. Cells were wished 2 times with PBS, and then treated with 10 µmol/L CCCP for 8 h prior to induce mitophagy. Cells were suspended into single cells and subsequently subjected to FCM to determine the flourecence intensity at 700 nm.

The Mitophagy detection kit (Cat.#MD01, Dojindo Molecular Technologies, Kumamoto, Japan) includes MtphagTracyker and LysoTracker for monitoring mitophagy. According to the manufacturer’s directions, cells were grown in confocal dishes and treated with MtphagTracyker and LysoTracker for 30 minutes. MtphagyTracyker (green) and LysoTracyker (red) were co-localized with mitophagy-indicating yellow dots. Fluorescence was measured three times for each group using a confocal fluorescence microscope (IX81-FV1000, Olympus, Markham, ON, Canada). Using NIH Image J, the fluorescence intensity of co-localized yellow dots was measured.

Samples were prepared with the Dojindo Molecular Technologies, Inc Mitophagy Detection Kit according to manufacturer’s instructions. Briefly, cells were incubated in a mitochondrial-binding dye prior to treatment. Three hours after treatment ceased, cells were incubated with a lysosome dye. Samples were analyzed and cell populations were quantified under the supervision of the UTHSCSA Flow Cytometry Core.