JC-1 MitoMP Detection Kit

Mitochondria synthesize ATP using oxygen to produce necessary energy for living cells. Lowering of mitochondrial activity and dysfunction are known to be closely related to cancer, aging, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Mitochondrial membrane potential is a parameter used to measure with mitochondrial condition.

How is the membrane potential detected?

JC-1 forms aggregate (in healthy mitochondria) with red fluorescence. As membrane potential decreases, JC-1 becomes monomers, which shows in green fluorescence. The change in ratio of red to green fluorescence is used as a indicator of mitochondrial condition.

---

Easy to Use

Easy to dissolve JC-1 has been difficult to dissolve, but this kit solves the problem!	Detect by Several Equipments Please refer to Data: Induced Apoptosis for experimental examples	Imaging Buffer Included HEPES included Imaging Buffer keeps the cell condition optimal for a long period

---

Procedure

---

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	[Plate Reader] Fluorescence intensity ratio 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	<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

1. Takushi Namba, “BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites”, Science Advances, 2019, DOI: 10.1126/sciadv.aaw1386.