Biochemistry
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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein
Chapters
Summary June 18th, 2020
This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.
Transcript
Current protocol assessors in textile, redox state changes in an intracellular compartment-specific manner. Our protocol enables a better understanding and monitoring all the physiological or pathophysiological mechanisms of oxidative stress. This technique allows non-disruptive quantification of the tiled eye sulfide ratio in intact cells using a ratio metric output to counterbalance the differences between signal expression levels, photo bleaching, and detection sensitivities.
This protocol can be applied to various organisms, including bacteria in plants. On day one of the experiment treat the cells from a confluent breast cancer cell line for two minutes, with two milliliters of 0.2, 5%trips in EDTA. When the cells begin to detach arrest the reaction with six milliliters of complete medium and sediment the cells by centrifugation.
Re suspend the pellet in five milliliters of fresh, complete medium, count the cells and dilute them to 1.5 times 10 to the fifth cells per one milliliter of complete medium. Then seed 1.5 times 10 to the fifth cells per milliliter of cells per well in a 6 well plate, and incubate the cells in the cell culture incubator overnight. Or add no viral row GFP transduction.
The next morning, add 12.5, 25, and 50 microliters of a one to 100 diluted six times 10 to the 10th plaque forming units per milliliter of add no viral erode GFP solution, in complete medium. Add that to the appropriate wells or the 6 well plate to transduce the cells with 50, 100, and 200 multiplicities of infection, respectively. When all of the sample Wells have been transduced with virus, return the cells to the cell culture incubator for 16 to 24 hours.
The next day, visualize the cells by fluorescence microscopy and replaced the supernatant with medium, without virus to allow the cells to recover for an additional 24 hours. To determine the optimal transduction efficiency, assess the fluorescence intensity and morphology of the cells by flow cytometry. Be sure to conduct all of the procedures that can cause infection from aerosols or splashes within a biosafety level two certified biological safety cabinet.
To assess the redox status of the cells, the next morning, incubate the cells and the appropriate Wells of the 6 well plate with 10 micromolar hydrogen peroxide for one hour, before detaching the cells with 750 microliters of 0.2 5%drips in EDTA per well, as demonstrated. Stop the reaction with two milliliters of complete medium per well and pull the supernatants for each condition in individual 15 milliliter conical tubes. Sediment the cells by centrifugation and wash the pellets in 500 microliters of PBS per well by centrifugations two times.
After the second wash, filter the cell suspensions through 40 micro meter meshes into flow cytometry compatible tubes on ice, protected from light. In the Flow cytometer software, run the zero multiplicity of infection control sample. Then analyze the hydrogen peroxide and untreated cells.
It will allow calculation of the mean fluorescent intensity ratio between the oxidized versus reduced forms of row GFP, using the formula as indicated. Here are the gating strategy for selecting a large number of cells by flow cytometry is shown. Flow cytometry can also be used to determine the dose response curve for row GFP analysis and the most efficient multiplicity of infection input.
According to the multiplicity of infection, dose response curve in this representative analysis, a 200 multiplicity of infection gave the highest road GFP expression, but the cell morphology was effected suggesting cytotoxicity. Therefore, the optimum transduction efficiency was determined to be a multiplicity of infection of 100. In this redox status analysis, oxidized and reduced row GFP mean fluorescence intensities were obtained from flow cytometry analysis for vehicle and four 10 micromolar positive control hydrogen peroxide treatments.
The overlaid histograms represent the shifts in the cell number in the 10 micromolar hydrogen peroxide and vehicle treated groups or reduced and oxidized erode GFP. Calculation of the ratio between the oxidized and reduced row GFP for this analysis revealed that 10 micromolar hydrogen peroxide caused a threefold increase in oxidation of row GFP compared to the vehicle treatment. Having accurate cell numbers is important for MRI calculations and reproducibility.
In order to have reproducible results, researchers should thoroughly mix the antiviral solutions to obtain homogeneous suspensions. This protocol allows researchers to study rapid redox changes as an integral part of the broad range of normal cellular processes and of disease progression in real time.
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