Analysis of mitochondrial transmembrane potential

Decrease in Avf»m is one of the early events of apoptosis (7, 8, 11, see also Chapter 8). Several types of membrane-permeable lipophilic cationic fluorochromes can serve as probes of Ai|>m in flow or laser-scanning cytometry. When live cells are incubated in their presence the probes accumulate in mitochondria and the extent of their uptake, measured by the intensity of the cellular fluorescence, is considered to reflect Ai|/m. Rhodamine 123 (Rhl23) and 3,3'-dihexiloxa-dicarbocyanine [DiOC6 (3)] are the fluorochromes most frequently used thus far to probe Av|<m (7, 8,11, 35). A combination of Rhl23 and propidium iodide (PI) was originally proposed as a cell viability assay discriminating between live cells that stain with Rhl23 only (green fluorescence), dead or dying cells whose plasma membrane integrity was compromised (cells with damaged plasma membrane, late apoptotic, and necrotic cells) that stain only with PI (red fluorescence), and early apoptotic cells that show somewhat increased stainability with PI but also stain with Rhl23 (35). The specificity of Rhl23 and DiOC6 (3) as mitochondrial probes is greater when these fluorochromes are used at low concentration. Still another probe of Ai|<m is the J-aggregate-forming lipophilic cationic fluorochrome 5,5',6,6'-tetrachloro-1,1',3,3'-tetrathylbenzimidazolcarbocyanine iodide (JC-1) (7). Its binding to mitochondria is detected by the shift in colour of fluorescence from green, which is characteristic of its monomeric form, to orange, which reflects its aggregation in mitochondria, driven by the transmembrane potential (7).

Measurement of mitochondrial transmembrane

Protocol 2.

potential

Reagents and stock solutions

• 3,3'-dihexiloxa-dicarbocyanirie [Di0C6 (3)1, rhodamine 123 and/or 5,5',6,6'-tetrachloro-1,1',3,3'-

tetraethylbenzimidazolcarbocyanine iodide (JC-1) (Molecular Probes)

• stock solution of Rh123: dissolve 0.1 mg of Rh123 in 1 ml distilled water

• stock solution of DiOC6 (3): dissolve 5 m-9 of DiOC6 (3) in 1.0 ml ethanol

• stock solution of JC-1: dissolve 1 mg JC-1 in 1 ml DMSO

• stock solution of PI: dissolve 1 mg PI in 1 ml distilled water

Each of the above stock solutions is stable and can be stored at 0-4°C in the dark for weeks.

• working solution of Di06 (3): add 5.0 m-I of the stock solution of Di06 (3) and 5 n-l of the stock solution of PI into 1 ml PBS

Methods

A. Staining with Rh 123 and PI and analysis by flow cytometry

1. Add 1.0 |xl of Rh123 stock solution to approximately 10® cells suspended in 1 ml of tissue culture medium (or HBSS) and incubate for 10 min at 37°C in the dark.

2. Add 20 |jul of the PI stock solution and keep for 5 min at room temperature in the dark.

3. Analyse cells by flow cytometry.

• use excitation in blue light (e.g. 488 nm line of the argon ion laser)

• use light scattering to trigger cell measurement

• measure green (Rh123) fluorescence at 530±20 nm

• measure red (PI) fluorescence at >600 nm

Analysis by LSC

To be analysed by LSC the cells have to be first attached to a microscope slide, then incubated with Rh123 and PI. To attach the cells:

1. Rinse the cells to be free of serum (or bovine serum albumin) and resuspend them in PBS to have 2 X 105-106 cells/ml.

2. Take 50 jjU of this suspension and deposit it within a shallow well on the microscope slide. The slides with wells can be made by preparing a strip of Parafilm 'M' (American National Can, Greenwich, CT) of the size of the slide, cutting a hole 2.0 cm x 0.5 cm in the middle of this strip, placing the strip on the microscope slide and heating the slide on a warm plate until the Parafilm starts to melt. Alternatively, the well can be made by drawing a border with a hydrophobic marker ('Isolator', Shandon Scientific, Pittsburgh, PA). The cell suspension is placed within the well on a slide and is maintained at room temperature at 100% humidity for 10 min to allow the cells to attach electrostatically to the slide surface.

3. The cells thus attached are then Incubated for 10 min with Rh123 and then with PI as described above for cells in suspension. A coverslip is then placed over the well and using illumination at 488 nm the cells' green and red fluorescence is measured by LSC.

4. If desired, the cells, while still attached to the slide after the measurement, can be fixed (e.g. in formaldehyde or ethanol) for subsequent staining with other dye(s) and/or visual inspection or measurement, following their relocation ('merge') on the slide.

B. Staining with DiOCe (3) and PI and analysis by flow cytometry

1. Suspend the cell pellet (—10® cells) in a working solution of DiOC6 (3).

2. Incubate for 15 min at room temperature in the dark.

3. Measure the cells' green and red fluorescence as described above for Rh123 and PI.

Analysis by LSC

Follow the procedure as described above for Rh123 and PI, except that after attaching the cells to the microscope slide incubate them with the working solution of DiOC6 (3) for 15 min.

C. Staining with JC-1 and analysis by flow cytometry

1. Suspend the cell pellet (~106 cells) in 1 ml of tissue culture medium with 10% serum.

2. Add 10 |j.l of the stock solution of JC-1. Vortex cells intensely during addition of JC-1 and for the next 20 sec.

3. Incubate cells for 10 min at room temperature in the dark.

4. Measure the cells' fluorescence by flow cytometry.

• excite fluorescence with blue light (488 nm line of argon laser)

• measure green fluorescence at 530±20 nm

• measure orange fluorescence at 570±20 or above (long pass filter) 570 nm

Analysis by LSC

1. Prepare the cells and incubate them with JC-1 in suspension as described above in steps 1-3.

2. Place 50 (jul of this suspension into a well on the microscope slide. Cover with a coverslip.

3. Analyse cells by LSC.

• use excitation in blue light (488 nm)

• use forward scattering as a contouring (triggering) parameter

• measure green fluorescence at 530±20 nm

• measure orange fluorescence at 570±20 or above (long pass filter) 570 nm

The decrease in Ai|;m which occurs during apoptosis can be measured by different markers, as discussed above, and shown in Figure 3. A combination of PI and DiOC6 (3) identifies non-apoptotic cells that stain only green, early apoptotic cells whose green fluorescence is markedly diminished, and late apoptotic or necrotic cells that stain with PI showing red fluorescence. Likewise, a combination of Rhl23 and PI labels live non-apoptotic cells green, early apoptotic cells dim green, and late apoptotic and necrotic cells red. The change in binding of JC-1 is manifested by a loss of orange fluorescence, which represents the aggregate binding of this dye in mitochondria. Green fluorescence of JC-1 also decreases during apoptosis, although to a lesser degree than orange. Because still another mitochondrial probe, nonyl acridine orange, reports mitochondrial mass but is not sensitive to Ai|jm (7) it is possible

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Figure3. Decrease of mitochondrial transmembrane potential during apoptosis measured by LSC. Apoptosis of U-937 cells was induced by their incubation with 5 ng/ml TNF-a combined with with 5 ji-g/ml of cycloheximide for 3 h; the cells were then incubated with DiOCe (3) and PI as described in Protocol 2. HL60 cells were treated with 0.15 CPT for 4 h to induce apoptosis and then stained with JC-1 as described in Protocol 2. The drop in mitochondrial transmembrane potential (A<(;m), which characterizes apoptotic cells, is reflected by a decrease in intensity of the green fluorescence of DiOC6 (3)-stained cells (CHX, top right panel) and loss of orange fluorescence of JC-1 stained cells (bottom right panel). Note that very few cells stain with PI (red fluorescence).

Figure3. Decrease of mitochondrial transmembrane potential during apoptosis measured by LSC. Apoptosis of U-937 cells was induced by their incubation with 5 ng/ml TNF-a combined with with 5 ji-g/ml of cycloheximide for 3 h; the cells were then incubated with DiOCe (3) and PI as described in Protocol 2. HL60 cells were treated with 0.15 CPT for 4 h to induce apoptosis and then stained with JC-1 as described in Protocol 2. The drop in mitochondrial transmembrane potential (A<(;m), which characterizes apoptotic cells, is reflected by a decrease in intensity of the green fluorescence of DiOC6 (3)-stained cells (CHX, top right panel) and loss of orange fluorescence of JC-1 stained cells (bottom right panel). Note that very few cells stain with PI (red fluorescence).

to analyse both the mitochondrial mass and Aipm in the same cells. Although apoptotic cells are sometimes more flattened on the slides and may have a greater diameter, their forward light scatter is diminished compared with the non-apoptotic cells.

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