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Procedure

Comparison of reagents for mitochondrial membrane potential detection

Data: Depolarization

HeLa cells treated with depolarizing reagent, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were stained with JC-1 MitoMP Detection Kit. Red fluorescence indicates normal membrane potential or health mitochondria. Untreated cells showed red fluorescence, while FCCP treated cells showed little red fluorescence.

Experimental Condition JC-1 concentration: 2 μmol/l in MEM, staining time: 30 min FCCP concentration: 100 μmol/l, FCCP treatment time: 1 h Imaging Condition Green: Ex 488 nm / Em 500-550 nm Red: Ex 561 nm / Em 560-610 nm Scale Bar: 20 μm

Data: Induced Apoptosis

Jurkat cells treated by apoptosis inducing reagent, Staurosporine, were stained with JC-1 MitoMP Detection Kit. Procedures for these data can be found in the Technical Manual.

[Fluorescence Microscope]
Fluorescence imaging of mitochondrial membrane potential in Jurkat cells

Imaging Condition Green: Ex 488 nm / Em 500-550 nm Red: Ex 561 nm / Em 560-610 nm Scale Bar: 80 μm

[Flow Cytometry]
Flow cytometric analysis of mitochondrial membrane potential in Jurkat cells

Detecting Condition Green: Ex 488 nm / Em 515-545 nm Red: Ex 488 nm / Em 564-604 nm

Plate Reader
Fluorescence intensity ratio of mitochondrial membrane potential in Jurkat cells

Detecting Condition Green: Ex 485 nm / Em 525-545 nm Red: Ex 535 nm / Em 585-605 nm

Required amount of Imaging Buffer solution by vessel type

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:
Mitophagy was not detected in untreated cells and the membrane potential was normal. However, reduction of membrane potential and mitophagy were observed in treated cells.

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