Cell Biology

The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.

Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death. In this protocol, we describe the histochemical detection of hydrogen peroxide (H₂O₂) and superoxide (O₂^-) anion, two of the most important ROS, in Brassica juncea seedlings by using 3,3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate. DAB is oxidized by H₂O₂ in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O₂^- to form a dark blue insoluble formazan compound. The protocol can be used in other plant species and for different plant tissues.

Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants systems. In several diseases (cancer, neurodegenerative diseases, cardiovascular diseases), the Redox balance is disturbed. In fact, excessive amounts of ROS can damage proteins, lipids and DNA at cellular level.

The choose of a sensitive method for detection of intracellular ROS is very important for detecting the disturbed Redox balance in pathological cells and after exposition of cells to different genotoxic agents (Irradiation, Oxidative stress, etc).

The detection of ROS in biological systems is difficult for several reasons: Method sensibility and probe specificity. The 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) fluorescent probe is commonly employed and may react with several ROS including hydrogen peroxide, hydroxyl radicals and peroxynitrite. The cell-permeant H2DCFDA passively diffuses into cells and is retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by ROS, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). The chloromethyl derivative of H2DCFDA (CM-H2DCFDA) provides much better retention in live cells than H2DCFDA. Dead or dying cells produces ROS. When we want to detect ROS in living cells, we have to stain cells by propidium iodide (PI) and evaluate ROS production only in living cells which are PI negative. In fact, PI intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. PI is excited at 488 nm and emits at a maximum wavelength of 617 nm. Because of these spectral characteristics, PI can be used in combination with other fluorescent probe such as CM-H2DCFDA (excitation/emission: 492–495/517–527 nm).

A probe fluorescence emission can be assessed by Flow cytometry, a standard fluorometer or fluorescence microscopy using appropriate filter.

Flow cytometry is commonly employed to detect intracellular ROS production. Flow cytometry measures fluorescence per cell. The cells is excited by the light source and emitted light from cells are converted to electrical pulses by optical detectors. Emitted Light is send to different detectors by using optical filters: 525 nm Band Pass Filter for FL-1 and 620 nm Band Pass Filter for FL-3. A 525 nm band pass filter (FL-1) placed in the light path prior to the detector will only allow “green” light into the detector. So, FL-1 is used in our protocol to collect green light corresponding to the oxidation of dichlorodihydrofluorescein (DCF) by ROS. FL-1 is the Green (FL-1) channel on flow cytometers. 620 nm Band Pass Filter (FL-3) only allow “red” light into the detector. Red fluorescence emission is measured in the Red (FL-3) channel on most flow cytometers.

The production of hydrogen peroxide (H₂O₂) has been recognized as an important feature of plant cells that undergo programmed cell death (PCD) during host-pathogen interaction. Thordal-Christensen et al. (1997) first described a method using chemical 3,3-diaminobenzidine (DAB) to detect the presence and distribution of H₂O₂ in barley leaves challenged by the powdery mildew fungus (Thordal-Christensen et al., 1997). Since then, this method has been adapted to many other plant species for in situ detection of H₂O₂. Here, we describe a modified protocol to stain and visualize H₂O₂ production in wheat leaves during infection by the necrotrophic fungus, Stagonospora nodrum or infiltration by the necrotrophic effectors produced by the fungus. The short version of this method has been reported in (Liu et al. 2012).