**1.1 Tissues and cell culture**

Fragments of the longitudinal smooth muscle layer of the guinea pig ileum (LSMLGPI) were prepared as described previously [20, 21], and the IR exposure in tissue fragments and confluent cell cultures from the LSMLGPI were exposed to single dose of 10–50 Gy, emitted by a 60Co γ-source [25]. The samples were radiated with a total dose of 10–50 Gy, and were then maintained for 3 days in Dulbecco's Modified Eagle Medium (DMEM).

#### **1.2 Colourimetry**

The ROS level was measured in the homogenates using the fluorescent method described by Yagi [26].

The H2O2-induced lipid peroxidation (LP) was measured through the oxidation of Fe2+ in the presence of xylenol orange in a spectrophotometer [27].

#### **1.3 Immunofluorescence analysis**


**55**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

were incubated with glutathione (GSH), 10<sup>−</sup><sup>3</sup>

stained as described in section *b* [29].

content, according to Traganos [31].

software as described by Gong [33].

**1.4 Western blot analysis**

**1.5 Confocal microscopy**

different periods of time.

**1.6 Electron micrography**

**1.7 Fluorescence microscopy**

JEOL, Tokyo, Japan).

**1.8 Statistical analysis**

c.To test if the generation of ROS contributes to apoptosis, some cultured cells

d.The generation of H2O2 was measured with 2′7′-dichlorofluorescein diacetate (DCFH-DA, as described by Hasui [30]) in live cultured cells. The cells were suspended in PBS, mixed with 0.3 mM DCFH-DA at 37°C to allow the conver-

e.To measure the degree of unbalanced growth, cultured cells were detached and stained with acridine orange (AO) for the evaluation of the ratio of RNA

f. The proteins involved in apoptosis were measured by immunofluorescence by specific antibodies, anti-: caspase 3, cyclin A, cyclin B2, cyclin E, PKCα, PKCε,

g.The cyclins A, B2, and E, and the DNA content were analyzed by MODFIT 3.0

The experimental procedure was performed as previously described [21] using LSMLGPI homogenates. The following antibodies were used, anti-: caspase 3, cyclin

LSMLGPI cells were seeded onto glass coverslips and exposed to IR. The mito-

For analysis of the KΨmito, 0.5 μM DiOC6(3) was used in DMEM, in vivo. The fluorescence was measured between 546/500 nm. To confirm the mitochondrial accumulation of DiOC6(3), the cells were incubated with KΨmito inhibitors [34] for

The cells were seeded as described by Claro [21], and were then radiated and fixed before being analyzed with a transmission electron microscope (1200 EXII,

Living cells were incubated with 2 μg/ml bisbenzimides diluted in DMEM and

Differences between irradiated and nonradiated groups were identified using the analysis of variance (ANOVA) of the unpaired Newman-Keuls tests (GraphPad

were analyzed between 461 and 350 nm, for DNA labeling.

Prism 5 software). Statistical significance was set at *P* < 0.05.

chondria were stained with a probe as described by Claro [20] in living cells.

TNFα, BAX, cytochrome c, BAG-1, BCL-2, and BCL-xL ([20, 32]).

A and cyclin B2, cyclin E, BCL-xL, BAX, cytochrome c, and BCL-2.

sion to DCF, and analyzed at 570/530 nm in the FL-1 channel.

yeast glutathione reductase type II (0.08 units/mg protein) and then fixed and

M reduced glutathione, and

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

