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Figure 1. Depending on the membrane polarization, JC-1 fluorescent probe can form monomers (FL-1) or J-aggregates (FL-2). Hence, Ai|im depolarization can be monitored by measuring the fluorescence in FL-1 and/or FL-2. (a) Ai|im was measured in untreated DU145 prostate cancer cells (shaded lire) and in cells treated with camptothecin (100 ng/ml, 24 h) (solid line), as described in Protocol 7. An increase in fluorescence (FL-1) is indicative of membrane depolarization, (bl Treatment of HL60 cells (human promyelocytic leukaemia cell line) with 5.0 ^g/ml camptothecin for 6 h causes a significant decrease in the fluorescence measured in FL-2 (seen as a shift to the bottom left panel in the contour plot), which is also indicative of di|fm disruption.

chondrial membrane results, in some instances, in redistribution of cytochrome c from the intermembrane space to the cytosol, followed by subsequent inner mitochondrial membrane depolarization.

Disruption of At|/m is believed to occur through permeability transition (PT), which can be activated in response to a variety of pro-apoptotic signal transduction molecules. The mitochondrial PT pore is a megachannel thought to be directly regulated by the Bcl-2/Bax complex (14). Opening of the PT pore, following PT, allows the release of solutes 1.5 kDa and smaller, and subsequent disruption of Av|im. As a consequence of intermembrane protein release, caspases and nucleases are released (14). Importantly, inhibitors of PT also inhibit apoptosis in several models of apoptosis, suggesting that disruption of mitochondrial function is of key importance to the apoptotic process (9).

The cell-permeant fluorescent probe 5,5',6,6'-tetrachloro-l,l',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) can be employed to monitor changes in Ai|;m in cells, by flow cytometry. In the presence of a high Ai|im JC-1 forms what are termed J-aggregates which fluoresce strongly at 590 nm (FL-2). Reduced A«j/m results in an increased FL-1 signal and/or in a reduced FL-2 signal in JC-l-stained cells (see Figure 1). This method allows subpopulations of cells with different mitochondrial properties to be identified (15).

Protocol 1. Measurement of mitochondrial transmembrane potential (AiJ>m) using JC-1

Equipment and reagents

• JC-1 (Molecular Probes), made as a • FACScan flow cytometer (e.g. Becton 5 mg/ml stock in DMSO (Sigma). Protect Dickinson), with an excitation source of from light and store at -20°C 488 nm

Method

1. Plate the cells in a standard 24-well plate at a density of 5 x 105/ml.

2. Treat the cells with the apoptosis-inducing agents either before, after, or during the incubation period, depending on the time point at which Ai|)m is to be measured.

3. Transfer the sample into a standard FACS tube.

4. Incubate the cells with 5 ^g/ml JC-1 for 15 min at 37°C, in the dark.

5. Collect the fluorescence emission through a 530/30 band pass filter (FL-1) and through a 585/42 band pass filter (FL-2), both on a log scale, using a FACScan flow cytometer (see Figure 1). For each sample, collect 5000-10000 events.

3. Detection of intracellular reactive oxygen intermediates (ROI)

Oxidative stress has been suggested as a possible common mediator of apoptosis in response to a number of stimuli (6, 8). Apoptosis can be induced by several agents that are known to cause oxidative stress, or it can be caused directly by the addition of oxidants (7). Accordingly, addition of antioxidants has been shown to block apoptosis induced by a variety of agents and to function at an early stage in the apoptotic process (6). Interestingly. Bcl-2 has been shown to act as an antioxidant (16,17).

The formation of ROI results from the reduction of oxygen. These ROI include hydrogen peroxide (H202), superoxide anion (02* "), and hydroxyl radical (OH*) (reviewed in ref. 10). Both superoxide and hydrogen peroxide are relatively unreactive compared with hydroxyl radicals, which can cause damage to most biological molecules. Possible sources of intracellular ROI include the depiction of cellular antioxidants such as GSH, disruption of mitochondrial respiration, and the activation of oxidant-producing enzymes such as NADPH oxidase.

Free radicals can activate tyrosine kinases (18). induce calcium release (19), cause sphingomyelin hydrolysis with consequent ceramide production (20), increase proteolytic degradation of proteins (21), and affect a variety of redox-sensitive targets such as transcription factors (22). Hence, oxidative stress can lead to the activation of other signal transduction pathways.

The fluorescent probes 2',7'-dichlorofluorescin diacetate (DCFH/DA) and dihydroethidium (DHE) may be used for the measurement of intracellular

Figure2. Peroxide levels were assessed in untreated HL60 cells (shaded line) and in cells treated with 5 jj,g/ml camptothecin for 15 min (dotted line), or cells treated with 1 mM H202 (broken line) for 30 min, as described in Protocol 2. After treatment with camptothecin or with H202 there is an increase in peroxide production which can be seen as a shift to the right in relative fluorescence (FL-1).

Figure2. Peroxide levels were assessed in untreated HL60 cells (shaded line) and in cells treated with 5 jj,g/ml camptothecin for 15 min (dotted line), or cells treated with 1 mM H202 (broken line) for 30 min, as described in Protocol 2. After treatment with camptothecin or with H202 there is an increase in peroxide production which can be seen as a shift to the right in relative fluorescence (FL-1).

Figure3. Superoxide levels were assessed in heat-shocked (43°C, 1 h) HL60 cells (dotted line) and in non-heat-shocked cells (shaded line), as described in Protocol 2, Heat shock caused an increase in superoxide anion levels in HL60 which can be seen as a shift to the right in relative fluorescence (FL-2).

peroxide and superoxide anion levels, respectively. Both probes can be used in a flow cytometer with a 15 milliwatt, air-cooled, argon ion laser. DCFH/DA is cell permeant and is non-fluorescent until the acetate groups are removed by cellular esterase activity and a peroxide group is subsequently encountered. Hydrolysed, oxidized DFCH/DA fluoresces at 529 nm (FL-1, log scale) (sec Figure 2) and is unable to leave the cell, thus allowing the measurement of intracellular peroxides by flow cytometry.

DHE is also cell permeant and is oxidized to ethidium by superoxide anion. Once oxidized, ethidium is free to intercalate with DNA in the nucleus, whereupon it emits fluorescence at 605 nm (FL-2; log scale) (see Figure 3).

Protocol 2. Measurement of intracellular peroxide levels Equipment and reagents

• DCFH/DA (Molecular Probes) prepared as a • FACScan flow cytometer (e.g. Becton 5 mM stock in DMSO (Sigmal. Protect from Dickinson), with an excitation source of light and store at -20°C 4B8 rim

Method

1. Plate the cells in a standard 24-well plate at a density of 5 x 105/ml.

2. Treat the cells with the apoptosis-inducing agents either before, after, or during the incubation period, depending on the time point at which peroxide levels are to be assessed.8

3. Transfer the sample into a standard FACS tube.

4. Incubate the cells with 5 jxM DCFH/DA, for 1 h at 37°C, in the dark.

Ana P. Costa-Pereira and Thomas G. Cotter Protocol 2. Continued

5. Assess the peroxide levels using a FACScan flow cytometer with excitation and emission settings of 488 nm and 530 nm, respectively (FL-1, log scale) (see Figure 2). For each sample collect 5000-10000 events.

"As a positive control for peroxide production, cells can be treated with 1 mM H202 for 30-60 min.

Protocol 3. Assessment of intracellular superoxide anion

Equipment and reagents

• DHE (Molecular Probes) prepared as a 10 • FACScan flow cytometer (e.g. Becton mM stock in DMSO (Sigma). Protect from Dickinson), with an excitation source of light and store at -20°C 488 nm

Method

1. Plate the cells in a standard 24-well plate at a density of 5 x 105/ml.

2. Treat the cells with the apoptosis-inducing agents either before, after, or during the incubation period, depending on the time point at which the levels of superoxide anion are to be assessed.

3. Transfer the sample into a standard FACS tube.

4. Incubate the cells with 10 jtM DHE, for 15 min at 37°C, in the dark.

5. Assess the superoxide levels using a FACScan flow cytometer with excitation and emission settings of 488 nm and 600 nm, respectively (FL-2; log scale) (see Figure 3). For each sample collect 5000-10000 events.

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