Comparison with Autophagy Marker

CCCP(carbonyl cyanide m-chlorophenyl hydrazone) has been added to normal and Parkin expressed cells. The strong fluorescence was not observed in normal HeLa cells(A)(B). On the other hand, the trong fluorescence was shown in Parkin expressed cells in 18 hours after additon of CCCP(C). Some of the puncta are co-localized with the autophagy marker(GFP-LC3). In addition, suppressed fluorescence of Mtphagy dye was observed when autophagy inhibitor, bafilomycin was added to Parking expressed cells(D). Because lysosomal pH was increased by the additon of bafilomycin.

Mitophagy Detection in Starved Cells

Living HeLa cells are co-stained with Mtphagy Dye and Lyso Dye under mitophagy induced condition.

Mtphagy Dye was added to HeLa cells and the cells were incubated for 6 hours under starved condition. Before observation of the cells, lysosomal staining dye, Lyso Dye was added to HeLa cells. The fluorescent intensity of Mtphagy Dye was increased in the starved HeLa cells but not in normal cells. In addition, Mtphagy Dye was co-localized with Lyso Dye in the starved cells.

Experimental Example: Induction of Mitophagy by CCCP

Induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial-uncoupling reagent with Parkin expressed HeLa cells

HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37^oC overnight in a 5%-CO2 incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent (Code#:H357), and incubated at 37^oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37^oC for 30 minutes with 250 μl of 100 nmol/l Mtphagy Dye working solution containing 100 nmol/l MitoBright Deep Red (※). After the washing of the cells with Hanks’ HEPES buffer twice, the culture medium containing 10 μmol/l CCCP was added to the well. After 24 hours incubation, mitophagy was observed by a fluorescence microscopy. After removing the supernatant, 250 μl of 1 μmol/l Lyso Dye working solution were added to the cells and incubated at 37^oC for 30 minutes. The cells were washed with Hanks’ HEPES buffer twice and then co-localization of Mtphagy, Lyso Dye and MitoBright Deep Red was observed by confocal fluorescence microscopy.

Observation of mitophagy using Parkin
expressed HeLa cells (upper panel) and normal HeLa cells (Lower)
A, E) Fluorescent images of Mtphagy Dye; B, F) Fluorescent images of Lyso Dye;
C, G) Fluorescent images of MitoBright Deep Red; D, H) Merged images

※Our sincere apology that we discontinued distributing MitoBright Deep Red.
     Please refer to MitoBright LT Deep Red (Code#:MT12) which improved retention ability. 

Mitophagy Induction and Detection of Mitochondrial Membrane Potential Changes

Mitochondrial condition in the carbonyl cyanide m-chlorophenyl hydrazine (CCCP) treated Parkin-expressing HeLa cells was compared with untreated cells using Mitophagy Detection Kit (MD01, MT02) and JC-1 MitoMP Detection Kit (MT09).

Result:

As a result, mitophagy was hardly detected in the CCCP-untreated cells, and mitochondrial membrane potential was maintained normally.

On the other hand, in CCCP-treated cells, we observed a decrease in mitochondrial membrane potential (decrease in red fluorescence of JC-1)

and induction of mitophagy (increase in fluorescence of Mtphagy Dye).

Experimental Condition
Transfection of Parkin plasmid to HeLa cells
– HileyMax (H357) was used to transfect Parkin plasmid to HeLa cells (Parkin plasmid/HilyMax reagent: 0.1μg/0.2 μL) by incubating overnight.
Detection of Mitophagy
1. Add 0.1 μmol/L Mtphagy working solution to Parkin expressing HeLa cells and incubated for 30 minutes at 37 ℃
2. Wash cells with HBSS
3. Add 10 μg/mL CCCP/MEM solution and inclubate for 2 hours at 37 ℃
4. Observe under fluorescence microscope
Detection of Mitochondrial Membrane Potential
1. Add 10 μg/mL CCCP/MEM solution to Parkin expressing HeLa cells and incubate for 1.5 hours at 37 ℃
2. Add 4 μmol/L JC-1 working solution (final concentration: 2 μmol/L) and incubate for 30 minutes at 37 ℃
3. Wash with HBSS and add Imaging Buffer Solution.
4. Observe under fluorescence microscope

Detecting Condition:

Mitophagy Detection
Ex: 561 nm, Em: 570-700 nm

Mitochondrial Membrane Potential Detection
Green Ex: 488 nm, Em: 500-550 nm
Red Ex: 561 nm, Em: 560-610 nm

Application

FCM

Mitochondrial contribution to lipofuscin formation

Procedure (Adherent cell):

1.Cells were washed twice with DMEM and afterwards incubated at 37 °C for 30 min with 100 nmol Mtphagy Dye diluted in DMEM.

2.After this incubation cells were again washed twice with DMEM followed by the addition of complete DMEM.

3.The induction of mitophagy was then accomplished by the addition of 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 24 h. Subsequently, fibroblasts were trypsinized and fluorescence intensity of Mtphagy Dye was measured by flow cytometry at 488 nm excitation and 655–730 nm emission.

Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy

Procedure (Suspension cell):

Briefly, 2×10⁶ CD4+ T cells were divided into four tubes containing serum-free RPMI.
Tube #1 was for unstained cells that followed all washing and media changing processes.
100 nmol/l Mtphagy Dye working solution was added to tubes #2, 3, 4 and then all tubes were incubated at 37°C for 30 minutes.
The cells were then washed with serum-free medium. After discarding the supernatant, complete medium (RPMI 1640, 10% FBS, 1% P/S/G) was added to all the tubes.
Ten μmol/l CCCP (Sigma-Aldrich) mitophagy-inducer was then added to tube #3, 100 nmol/l Bafilomycin A1 (Sigma-Aldrich) autophagy inhibitor was added to tube #4.
All tubes were then incubated at 37 °C for 18 hours.
After 18 hours, cells were washed with FACS buffer.
Mtphagy Dye fluorescence detection by flow cytometry (BD FACSCANTO II) was performed using 488 nm for excitation and 695 nm for emission (this corresponds to the PerCP cy5.5 channel). Data were analyzed using FLOWJO software (version 10).

Microplate Reader

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: involvement of ROS and mitophagy