Mitochondria Research for Mammalian Cells

∼ Features ∼

  • Beginner Friendly
    • Easy to Dissolve
    • Imaging Buffer optimize Cell Condition for Measurements

 


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


JC-1 MitoMP Detection Kit (MT09) and Mitophagy Detection Kit (MD01) are useful tools for evaluation of mitochondrial homeostasis

The effect of ER membrane protein BAP31 in mitochondrial homeostasis was evaluated using our JC-1 MitoMP Detection Kit (MT09) and Mitophagy Detection Kit (MD01). JC-1 was used for fluorescence imaging and microplate reader assay. JC-1 monomers (green) in U2OS cells treated with
siBAP31 was increased. The ration of JC-1 aggregates (red) / JC-1 monomers (green) was decreased. Loss of BAP31 decreases mitochondrial transmembrane electric potential.
Whether or not mitophagy is induced by loss of BAP31 was evaluated using Mitophagy Detection Kit (MD01). FCCP with oligomycin was used as mitophagy inducer. Loss of BAP31 induced mitophagy but was a minor effect compared with mitophagy inducer–treated cells.

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


T. Nechiporuk, et al. identified that TP53, BAX, and PMAIP1 were key genes in Venetoclax resistance in acute myeloid leukemia. In their research, our JC-1 MitoMP Detection Kit (MT09) and Mitophagy Detection Kit (MD01) were used to evaluate mitochondrial stress by influence of gene. The cells that inactivated TP53 and BAX were treated with Venetoclax and CCCP. It was confirmed by JC-1 that TP53-mutant and BAX-mutant cells treated with CCCP didn’t lose mitochondrial membrane potential using flow cytometry. Mitophagy by CCCP treatment was less influence in TP53-inactivated and BAX-inactivated cells.

2) T.Nechiporuk, et al., "The TP53 Apoptotic Network Is a Primary Mediator of Resistance to BCL2 Inhibition in AML Cells", Cancer Discov., 2019, 9, 910-25.

Measurement Example

Doxorubicin (DOX) acts to inhibit cell proliferation during G2/M phases of the cell cycle and induces cellular senescence. After adding DOX to A549 cells, higher histogram peaks for the G2/M phase (Cell Cycle Assay Solution Blue and Deep Red), induces cellular senescence (Cellular Senescence Detection Kit - SPiDER-βGal), and the differences in mitochondrial membrane potential (JC-1 MitoMP Detection Kit) were observed.


Storage Condition: 0-5 oC and protect from light

Shipping Condition: ambient temperature

Reference:

1) Takushi Namba, “BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites”, Science Advances, 2019, DOI: 10.1126/sciadv.aaw1386.
2) I. Kawahata, L. Luc Bousset, R. Melki and K. Fukunaga , "Fatty Acid-Binding Protein 3 is Critical for α-Synuclein Uptake and MPP+-Induced Mitochondrial Dysfunction in Cultured Dopaminergic Neurons ", Int J Mol Sci., 2019, 20, 5358.
3) T.Nechiporuk, et al., "The TP53 Apoptotic Network Is a Primary Mediator of Resistance to BCL2 Inhibition in AML Cells", Cancer Discov., 2019, 9, 910-25.
4)M. Kurano, K. Tsukamoto, T. Shimizu, H. Kassai, K. Nakao, A. Aiba, M. Hara and Yatomi , "Protection Against Insulin Resistance by Apolipoprotein M/Sphingosine 1-Phosphate ", Diabetes, 2020, DOI: 10.2337/db19-0811.


Item # Description/Size Availability Qty Break Price Quantity
MT09-10
1 set
1-2 business days 1 $230.00

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