Loss of mitochondrial membrane potential is indicative of apoptosis. ICT’s MitoPT JC-1 Assay utilizes the potentiometric fluorescent dye JC-1 to detect mitochondrial membrane permeability and membrane depolarization.
The JC-1 dye has a delocalized positive charge dispersed throughout its molecular structure. In addition, its lipophilic solubility enables it to be readily membrane permeant and penetrate living cells. The JC-1 dye enters the negatively charge mitochondria where it accumulates and fluoresces orange upon excitation. When the mitochondrial membrane potential collapses in apoptotic cells, MitoPT JC-1 becomes distributed throughout the cytosol. Analyze your results using a flow cytometer, fluorescence plate reader, or fluorescence microscopy.
1. Prepare samples.
2. Create controls with CCCP.
3. Dilute 10X Assay Buffer 1:10 with diH2O.
4. Reconstitute MitoPT JC-1 with DMSO.
5. Dilute MitoPT JC-1 with 1X Assay Buffer.
6. Add MitoPT JC-1 to each sample.
7. Incubate 15-30 minutes.
8. Wash cells: add 1X Assay Buffer and spin cells (twice).
9. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT JC-1 excites at 488 nm. Aggregated MitoPT JC-1 (orange) emits at 590 nm, monomeric MitoPT JC-1 (green) emits at 527 nm.
Healthy cells fluoresce red, dying cells fluoresce green. Jurkat cells were stained with ICT’s MitoPT JC-1 dye and analyzed with a fluorescence microscope containing a long bandpass filter (excitation at 490 nm and emission >510 nm). In non-apoptotic healthy cells (2 cells at left), the reagent aggregates inside intact mitochondria and fluoresces red. As the mitochondrial membrane potential drops and cells enter apoptosis, the dye disperses throughout the cell. The reagent then assumes a monomeric form and fluoresces green (3 cells on right).
Using a flow cytometer to analyze cells labeled with MitoPT JC-1, the instrument will measure apoptosis by monitoring the amount of red fluorescence in each region. Healthy cells with intact mitochondria fluoresce red due to aggregated MitoPT JC-1 and appear in R2. As the mitochondrial membrane potential collapses and cells enter apoptosis, MitoPT JC-1 reagent is dispersed throughout the cell, converting to its green fluorescent monomeric form. The amount of red fluorescence drops as these cells enter R3. In this example, Jurkat cells were either treated with DMSO (negative, non-induced cells) or with staurosporine (apoptotic, induced cells) for 4 hours and then labeled with MitoPT JC-1 for 15 minutes.
Using a fluorescence plate reader, healthy cells will give a high OD reading of red fluorescence; apoptotic cells will generate a lower reading of red fluorescence. In this example, Jurkat cells were either treated with DMSO (negative, non-induced cells) or with staurosporine (apoptotic, induced cells) for 4 hours and then labeled with MitoPT JC-1 for 15 minutes. As the mitochondrial membrane potential collapsed, indicating apoptosis, the amount of red fluorescence dropped by half (51%) in Jurkat cells from 167 RFU to 82.
• Reagent name: JC-1
• Target: Mitochondrial depolarization
• Excitation/Emission: 488 nm / 590 nm & 527 nm
• Method of Analysis: Flow Cytometry, Fluorescence Microscope, Fluorescence Plate Reader
• Types of Samples: Cell culture
• Storage: -20°C
• Shipping: Ships overnight (domestic), International Priority Shipping
Kit 924: 100 Tests
• MitoPT JC-1 Reagent, 100 Tests, #6261
• 10X Assay Buffer, 60 mL, #685
• CCCP, 50 mM, 125 µL, #6257
• Kit Manual
Kit 911: 400 Tests
• MitoPT JC-1 Reagent, 400 Tests, #6260
• 10X Assay Buffer, 2 x 125 mL, #6259
• CCCP, 50 mM, 600 µL, #6258
• Kit Manual
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