MitoProbe DiIC1(5) Mitochondrial Membrane Potential Protocol

Cationic cyanine dyes have been shown to accumulate in cells in response to membrane potential¹ and membrane potential changes have been studied in association with apoptosis.^2-3 The MitoProbe™ DiIC1(5) Assay Kit provides solutions of the cyanine dye DiIC1(5) (1,1′,3,3,3′,3′-hexamethylindodicarbo - cyanine iodide, Figure 1) and CCCP (carbonyl cyanide 3- chlorophenylhydrazone), for the study of mitochondrial membrane potential. DiIC1(5) penetrates the cytosol of eukaryotic cells. At concentrations below 100 nM, the dye accumulates primarily in mitochondria with active membrane potentials. DiIC1(5) stain intensity decreases when cells are treated with reagents that disrupt mitochondrial membrane potential, such as CCCP (Figure 2).

Figure 1. Structure of DiIC1(5), molecular weight: 510.46.

Figure 2. Decrease in DiIC1(5) fluorescence with the addition of CCCP. Jurkat cells were stained with 50 nM DiIC1(5) alone (black line) or in the presence of 50 μM CCCP (gray line).

Cells stained with DiIC1(5) can be visualized by flow cytometry with red excitation and far red emission. The reagent can be paired with other reagents, such as blue-excited propidium iodide and annexin V–Alexa Fluor 488 dye (Vybrant Apoptosis Assay Kit #2, V13241), for multiparametric study of vitality and apoptosis (Figure 3). Combining DiIC1(5) dye with an annexin Vconjugate results in superior resolution of subpopulations when compared to results obtained from other commonly used dyes.

Figure 3.  Camptothecin-treated Jurkat cells stained with DiIC1(5) and annexin V–Alexa Fluor 488 dye. Cells were incubated for 3 hours with 10 μM camptothecin at 37°C, 5% CO₂, then stained with DiIC1(5), annexin V–Alexa Fluor 488 dye, and propidium iodide. In Panel A, most of the annexin V–positive cells show reduced DiIC1(5) fluorescence. In Panel B, propidium iodide–positive cells show low to very low DiIC1(5) fluorescence.

Kit Contents

• DiIC1(5), 625 μL of 10 μM DiIC1(5) in DMSO
• CCCP, 125 μL of 50 mM CCCP in DMSO

Storage and Handling

Upon receipt, components should be stored at 2–6°C. DO NOT FREEZE. Before opening, each vial must be at room temperature. When stored properly, both the DiIC1(5) and CCCP solutions should be stable for at least twelve months.

Spectral Characteristics

The approximate excitation and emission peaks of DiIC1(5) are 638 nm and 658 nm, respectively. Cells labeled with DiIC1(5) can be analyzed by flow cytometry using 633 nm excitation and far red emission, and by fluorescence microscopy using standard filters for Alexa Fluor 633 dye.

The following protocol describes introducing DiIC1(5) reagent into the cultured cells and analyzing the stained cells by flow cytometry. Suggested initial conditions may require modifications because of differences in cell types and culture conditions. The concentration of probe for optimal staining will vary depending upon the application. A concentration range should be tested starting around 50 nM DiIC1(5). CCCP controls should be used to confirm that the DiIC1(5) response is sensitive to changes in membrane potential.

Labeling Cells with DiIC1(5)

Before beginning the experiment, ensure that the vials of DiIC1(5) and CCCP have equilibrated to room temperature.

1. For each sample, suspend cells in 1 mL warm medium, phosphate-buffered saline, or other buffer at approximately 1 x 10⁶ cells/mL.


2. For the control tube, add 1 μL of 50 mM CCCP (supplied with the kit, 50 μM final concentration) and incubate the cells at 37°C for 5 minutes. Note: CCCP can be added simultaneously with DiIC1(5). Titration of the CCCP may be required for optimal results with each cell type.


3. Add 5 μL of 10 μM DiIC1(5) (supplied with the kit, 50 nM final concentration) and incubate the cells at 37°C, 5% CO₂, for 15 to 30 minutes. If performing additional labeling, for example with an annexin V conjugate, follow the protocol below, beginning with step 1. If no additional staining is to be performed, proceed with step 4.


4. OPTIONAL: Wash cells once by adding 2 mL of warm phosphate-buffered saline (PBS) or other buffer to each tube of cells.


5. Pellet the cells by centrifugation.


6. Resuspend by gently flicking the tubes. Add 500 μL PBS (or other suitable buffer) to each tube.


7. Analyze on a flow cytometer with 633 nm excitation using emission filters appropriate for Alexa Fluor® 633 dye (Figure 2). Gate on the cells, excluding debris. Using the CCCP-treated sample, perform standard compensation.

Additional Labeling with an Annexin V Conjugate and Propidium Iodide

It is possible to label the DiIC1(5)-stained cells with other markers for apoptosis or viability, as long as the fluorescence emission of the additional label is spectrally resolved from DiOC1(5). The example below is a protocol for labeling with an annexin V–Alexa Fluor® 488 conjugate and propidium iodide.

1. After step 3 (above), wash cells once by adding 2 mL of warm phosphate-buffered saline or other buffer to each tube of cells.


2. Pellet the DiIC1(5)-stained cells and resuspend in 100 μL of 1X annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4).


3. Add 5 μL annexin V conjugate (e.g. annexin V–Alexa Fluor 488 conjugate, V13241).
   
   Note: 5 μL is appropriate for annexin V conjugates from Molecular Probes. Conjugates purchased from other suppliers may require a different volume to be effective.


4. Add 1 μL of a 100 μg/mL propidium iodide solution (V13241, prepared according to instructions accompanying that kit).


5. Incubate the samples at 37°C for 15 minutes. (37°C is important to maintain membrane potential.)


6. Add 400 μL annexin binding buffer.


7. Analyze on a flow cytometer with 488 nm and 633 nm excitation using emission filters appropriate for Alexa Fluor 488, R-phycoerythrin, and Alexa Fluor 633 dye (Figure 3).