---+ Measuring Intracellular Reactive Oxygen Species (ROS) This procedure is commonly utilized to quantify ROS. For more information about limitations associated with this assay and other methods to measure ROS, please read [[http://www.sciencedirect.com/science/article/pii/S0891584911006058?via%3Dihub][this helpful paper]]. This protocol was adapted from methods found [[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015979][in here]]. 1 Grow 5 mL overnight cultures of your favorite strains. 1 The following day, dilute back each culture 1:100 into fresh media. 1 Once the cells have reached mid-exponential phase (OD of ~0.5), pull the cultures out of the incubator and at this point you can add an ROS elicitor if desired. Some common ROS elicitors include potassium tellurite (K<sub>2</sub>TeO<sub>3</sub>), chromate (K<sub>2</sub>CrO<sub>4</sub>), and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Each of these compounds will induce the formation of different reactive oxygen species, so you will want to pick one (or all) that are suitable for your experiment. Additionally, it is good practice to measure ROS in the absence of any ROS elicitor. 1 For ROS induction, we commonly use 0.5μg/ml of K<sub>2</sub>TeO<sub>3</sub> and expose the cells for 30 minutes, shaking at 37°C. 1 Centrifuge cells at 3,000g for 5 minutes at 4°C, wash once with cold saline phosphate buffer (pH = 7.3), and resuspend in 2X the initial volume with phosphate buffer. * Recipe for the saline phosphate buffer = 137mM NaCl, 2.7mM KCl, 10mM Na<sub>2</sub>HPO<sub>4</sub>, 2mM KH<sub>2</sub>PO<sub>4</sub>, make sure to pH the buffer to 7.3 using NaOH 1 Next, you will need to expose the cells to the ROS fluorescent dye, 2',7'-dihydrodichlorofluorescein diacetate (100mM final concentration). Perform the incubation in a 37°C water bath for 60 minutes. 1 Centrifuge the cells at 3,000g for 5 minutes at 4°C, wash once with cold phosphate buffer, and resuspend washed cells in phosphate buffer. 1 The fluorescence can be measured in a plate reader or by flow cytometry, excitation = 490 and emission = 519.
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Topic revision: r4 - 2017-07-31 - DaciaLeon