How to prepare samples for Mitochondria Potential Assay

Background

Cell death can occur by two distinct mechanisms, necrosis or apoptosis. Necrosis occurs when cells are exposed to harsh physical or chemical stress (e.g., hypothermia, hypoxia) while apoptosis is a tightly controlled biochemical process by which cells are eliminated and where the cell is an active participant in its own termination (“cellular suicide”). 

Apoptosis is one of the main types of programmed cell death which occur in multicellular organisms and is characterized by a series of events that lead to a variety of morphological and biochemical changes, including membrane blebbing, cell shrinkage, alteration of membrane asymmetry and permeability, condensation of chromatin and nucleus, DNA fragmentation, and formation of membrane bound vesicles (apoptotic bodies) 

Reliable detection and monitoring of apoptosis is crucial for the development of treatments for apoptosis-associated diseases and for investigating apoptotic mechanisms in general. 

Loss of the mitochondrial membrane potential is known to precede apoptosis and chemical-hypoxia-induced necrosis. The lipophilic cationic dye, JC-1, display potential-dependent accumulation in the mitochondria and provides a simple, fluorescent-based method for distinguishing between healthy and apoptotic cells.

In healthy cells, the negative charge established by the intact mitochondrial membrane potential facilitates the accumulation of JC-1 in the mitochondrial matrix.

At high concentrations JC-1 forms aggregates and become red fluorescent. In apoptotic cells the mitochondrial potential collapses and JC-1 localizes to the cytosol in its monomeric green fluorescent form.

How to prepare samples for Mitochondria Potential Assay

Cells are stained with JC-1 and DAPI. Cellular JC-1 monomers and aggregates are detected as green and red fluorescence, respectively. Apoptosis and thereby also mitochondrial depolarization are revealed as a decrease in the red/green fluorescence intensity ratio. Necrotic and late apoptotic cells are detected as blue fluorescent (DAPI) cells.

  1. For each sample, suspend cells in 1 mL medium, PBS or other appropriate buffer at approximately 1×106 cells/mL. 

Note: Adjust volume as necessary to yield the above cell concentration, use only 1 mL of diluted sample.

  • Add 12.5 μL of Solution 7 (final concentration: 2.5 μg/mL) to the cell sample and incubate 10 minutes at 37⁰ C. 

Note: Duration of the staining may depend on the specific cell type. If necessary, incubation time can be extended to 30 minutes. 

  • Centrifuge stained cells at 400 for 5 minutes at room temperature and remove the supernatant completely without disturbing the cell pellet. 
  • Resuspend cell pellet in 1-2 mL PBS by pipetting, centrifuge at 400 for 5 minutes at room temperature and remove the supernatant completely without disturbing the cell pellet. 
  • Wash again. Resuspend cell pellet in 1-2 mL PBS by pipetting, centrifuge at 400 for 5 minutes at room temperature and remove the supernatant completely without disturbing the cell pellet. 

Note: Careful washing of the sample is crucial for achieving valid results. 

  • Resuspend cell pellet by pipetting in 250 μL Solution 8
  • Sample is ready for analysis. 
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