The aim of this experiment is to detect the presence of reactive oxygen species in cell cultures. This is accomplished by first detecting reactive oxygen species using a fluorescent dye. Next, the levels of nitric oxide in cells are measured using a colormetric assay.
Finally, the effect of reactive oxygen species is determined by detecting the reduction or oxidation of glutathione using a luminescent assay. My name is Danu. I'm a poster in the lab of Patrizia Yoda, and I will be demonstrating this procedure.
Carboxy H 2D CFDA is non fluorescent, but in the presence of reactive oxygen species, this reagent is oxidized and fluoresce fluoresces green immediately prior to use. Prepare a fresh stock solution of carboxy H 2D CFDA in sterile DMSO or 100%ethanol. Wash the cells with HEPs buffered salt solution or phosphate buffered saline to remove traces of the original medium.
In this assay, we use the human leukemia jerk cat cell line. After centrifuging dispose of the SUP natant load the cells with the dye as a final concentration of one micromolar in regular culture medium with reduced serum incubate the cultures for 30 minutes in the dark in a conventional incubator, discard any unused dye solution. Next, remove the Carbox CH 2D CFDA containing medium and wash the cells twice with HBSS or PBS.
Transfer the cells to a plate and from this step forward, protect the cells from light. Then add fresh medium containing a drug of choice to the carboxy H 2D CFDA loaded cells and incubators desired. In this example, we use 0.03%hydrogen peroxide for one hour.
Control cells include downloaded untreated cells and unstained untreated cells. Immediately assess ROS by analyzing the cells with flow cytometry. Using the FL one channel, using a fluorescence plate reader or by fluorescence microscopy using excitation and emission wavelengths that are appropriate for green fluorescence.
Before beginning this assay, prepare the following solutions. A 0.1%solution of one nyl ethylene diamine dihydrochloride or NED diluted in sterile water. A 1%solution of sulfuryl amide diluted in 5%phosphoric acid, and a serial dilution of sodium nitrite in sterile medium, beginning with a 0.1 molar standard stock solution store NED and Sulfuryl Aide Solutions in the dark of four degrees Celsius culture cells in a 96 well plate using triplicates for each condition and proper controls.
In this experiment to induce nitric oxide production mouse macrophage raw 2 6 4 0.7 cells are treated with 100 nanograms per milliliter of LIPA polysaccharide or LPS and recombinant IL four. To perform the assay, bring both reagents to room temperature, spin the plate, collect the cell supernatants, and transfer 50 microliters to a new 96 well plate. Then prepare limiting dilution wells of the standard stock solution.
To make a standard curve, add 50 microliters of sulfur IDE solution to each sample and control well and mix well. Incubate room temperature for 10 minutes in the dark. Next, add 50 microliters of NED solution to each sample and control well and mix.
Well incubate the plate in the dark for an additional 10 minutes With the grease assay, there's a rapid change of active oxygen species stitchers. Therefore, it's important to work fast to prevent losing the raw signal. Measure the absorbance immediately using a plate reader with filter wavelengths between 520 nanometers and 550 nanometers, a violet color will appear in the positive wells.
Compare the results with the standard to check the stability of the solutions the day before the assay seed cells in a 96 well white plate in triplicate for each condition the following day, treat the cells with the drug of choice for the required time. In this example, jca and a 5 49 cells are treated for one hour with five millimolar and 2.5 millimolar hydrogen peroxide respectively. Raw 2 6 4 0.7 cells are treated with 200 nanograms per milliliter of LPS for 16 hours.
Remove the medium from wells with adherence cells. Do not remove the medium if the cells are in suspension. No more than 30 minutes prior to performing the assay.
Prepare reduced and oxidized glutathione lysis reagents. Add the reagents to the desired wells and shake for five minutes. At room temperature, add the luciferian generation reagent and incubate for 30 minutes at room temperature.
Then add the luciferian detection reagent and incubate for an additional 15 minutes at room temperature. Finally, read the bioluminescence signal at an integration timer. 0.25 to one second per well using a plate, read luminometer here.
Jerk out cells treated with hydrogen peroxide are compared to non-treated cells. Reactive oxygen species induce the modification of carboxy H 2D CFDA that fluoresces green as detected by facts. The results confirm the presence of reactive oxygen species in treated cells.
In this example, raw 2 6 4 0.7 cells were treated with LPS and IL four. A significant increase in nitric oxide production was detected in treated cells compared to control untreated cells. Here, raw 2 64 0.7 cells were treated with LPS and jca, and a 5 49 cells were treated with hydrogen peroxide.
The results are expressed as the ratio of reduced to oxidized glutathione. Lower ratios for glutathione were detected in treated cells compared to control untreated cells indicating that proteins were more oxidized intreated cells. After watching this video, you should have a good understanding of how to detect raw using this assays.
