Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

The overall goal of the following experiment is to provide an easy and versatile solution to monitor various reactive oxygen species or ROS and their localization and provide further insights into ROS related cellular mechanisms. This is achieved by performing three different assays, the first of which uses RST green or OB G coated beads and live microscopy, ob g fluorescein, and Alexa. Fluor 594 are covalently coupled to the surface of three microns sica beads via BSA.

The fluorescence emission from the OBG fluorescein indicates the oxidization by ROS, while the signal from Alexa Fluor 594 helps localize the beads and serves as a control for fluorescence quantification. The second method utilizes the superoxide sensitive probe dihydro ethidium or DHE in a plate reader based assay. As a result of oxidation by superoxide ethidium is able to intercalate into nuclear and mitochondrial DNA and has shifted excitation and emission peaks at 522 nanometers and 605 nanometers respectively.

The fluorescence intensity of AUM corresponds to the amount of superoxide production, allowing quantitative measurement of intracellular superoxide generation in the cells. The third method is also a plate reader based assay. The hydrogen peroxide sensitive probe plex ultra red or a UR is used to quantitatively measure extracellular hydrogen peroxide production.

The fluorescent intensity of a UR OX corresponds to the amount of hydrogen peroxide produced outside of the cells. Results are obtained that show localization of dynamic ROS generation based on the O-B-G-D-H-E and a UR assays. The main advantage of this technique over the other existing methods such as plate rate based OB GSAs, is that it dynamically visualizes the Al Roth generation at the single cell level.

Instead of mirroring and averaged Roth signal from a population of cells. Such population averaging methods tend to obscure critical information due to non synchronous phagocytosis. To begin the procedure for coding beads with oxy, Burt Green or OBG, add one milliliter of a suspension of 3.0 micron carboxylated silica beads into a 1.5 milliliter tube.

Spin the tube quickly remove the supernatant. Add one milliliter of PBS and vortex in this manner. Wash the beads three times with PBS.

After the third wash, remove the supernatant and resuspend the beads. In one milliliter of PBS containing 25 milligrams per milliliter cyanide, the cyanide solution must be freshly prepared each time before use incubate on a wheel for 15 minutes. This will activate the carboxylated silica beads to covalently bind pre-labeled BSA after 15 minutes, centrifuge the tube, remove the supernatant.

Add one milliliter of coupling buffer and vortex wash three times with coupling buffer by a quick spin and vortexing to remove excess cyanide, mix the washed beads with 500 microliters of coupling buffer containing one milligram of OBGH two H-F-F-B-S-A and then fill the tube with nitrogen gas from a standard gas can. After capping the tube incubated on a wheel for 14 hours at room temperature in the dark on the following day, quench unreactive OBG by washing the beads twice with one milliliter of quenching buffer by a quick spin and vortexing then wash twice with coupling buffer to remove the quenching buffer. After removal of the supernatant, add one milliliter of coupling buffer containing 50 micrograms of LOR 594 cin a middle ester to conjugate to BSA.

Fill the tube with nitrogen cap and incubate on a wheel for 1.5 hours at room temperature in the dark. At the end of the 1.5 hour incubation, stop the reaction by washing the beads three times with one milliliter of quenching buffer. This is followed by washing the beads three times with one milliliter of PBS.

Finally, Ray, suspend the beads in one milliliter of PBS with two microliters of 10%weight per volume. Azide for long-term storage. Measure the concentration of beads in the suspension with a hemo cytometer, it is typically one to two times 10 to the ninth beads per milliliter.

Fill the tube with nitrogen cap and store it four degrees Celsius in the dark. Start this procedure by harvesting exponentially growing alium cells from a 10 centimeter Petri dish plate. Different densities of cells on three centimeter dishes with an optically clear plastic or glass bottom and grow them overnight on the following morning.

Choose the dishes that are about 80%confluent for use in the experiment. Replace the culture medium with low flow or LF medium and incubate for two hours before the experiment in order to decrease the extracellular and intra endosomal autofluorescence of the HL five C culture medium. Melt 10 milliliters of 1.5%BDO agar in LF medium, and pour the agar onto a flat surface in order to form an agar layer about one millimeter thick.

Wait for 10 to 15 minutes for the agar to solidify. Cut the agar layer into two by two centimeter squares and place in LF medium for later.Use. Repair the widefield microscope.

Set the temperature of the environmental chamber to 22 degrees Celsius and adjust the settings required for the experiment. After two hours of incubation, aspirate the LF medium from the three centimeter dish, but leave the cell monolayer covered by a thin film of medium. Dilute the previously prepared OBG coated beads to 1.5 times 10 to the seventh beads per milliliter and add 10 microliters onto the cell layer.

Take one square agar sheet and drain any excess liquid, but keep the agar wet. Gently put the agar square on top of the cell layer. The agar overlay increases contact between beads and cells, thereby improving uptake.

It also slightly compresses the cells, keeping them better in the focal plane of the objective. Place the lid onto the dish For the success of the experiment. Don't move the AGA overlay when it is covering on the cells and also it is critical to start the imaging step immediately.

Next, place the dish on the microscope stage and automatically take pictures in the red, green and phase channels every minute for two hours or longer. To measure ROS production, select and focus on cellular events that contain the whole process of phagocytosis. Using professional image processing software, merge the optimized three channels and assemble the pictures into a movie.

Quantify fluorescence intensities of each of the selected beads in the red and green channels. The ratio of red to green will reflect the dynamic FOMO R os production of the cells. To measure intracellular superoxide production first, collect 180%confluent dish of alium in 10 milliliters of HL five C medium next centrifuge alium cells at 850 times G for five minutes and carefully and completely aspirate the medium resus resuspend cells in SS 6.4 buffer count cells and dilute to a final density of six times 10 to the six cells per milliliter.

Add 50 microliters of cell suspension into each well of a white non-transparent 96 well plate dilute dihydro ethidium or DHE stock 500 fold with SS 6.4 buffer. And then use a multi-channel pipette to dispense 50 microliters of the diluted DHE into each well of the 96 well plate. The final concentration of DHE is 30 micromolar.

The reaction will start immediately. Stimuli or inhibitors can be added at this point or at other time points according to specific needs. Incubate the cells with medium shaking at 22 degrees Celsius.

Read fluorescence with a fluorescence microplate reader every two minutes for one hour. Use the endpoint top reading mode with fluorescence excitation at 522 nanometers and emission at 605 nanometers. Begin this procedure by preparing a 96 well plate with dium cells as demonstrated in the previous segment.

Prepare diluted horse radish peroxidase or HRP by adding five microliters of HRP stock into 10 milliliters of SS 6.4 buffer. Invert the tube to mix well and keep on ice. The diluted HRP solution is at 0.05 units per milliliter.

Prepare the plex ultra red or a UR reaction mixture by mixing the a UR stock and diluted HRP solution into SS 6.4 buffer to final concentrations of 6.25. Micromolar a UR and 0.005 units per milliliter. HRP add 50 microliters of a UR mixture into each well.

The reaction will start immediately. Stimuli or inhibitors can be added at this point or at other time points according to specific needs. Incubate the cells with medium shaking at 22 degrees Celsius.

Read fluorescence with a fluorescence microplate reader every two minutes for one hour. Use the endpoint top reading mode with fluorescence excitation at 530 nanometers and emission at 590 nanometers. The coating efficiency of the OB g fluorescein is tested by using hydrogen peroxide and HRP to oxidize the coated beads in vitro and checking the emission spectrum using excitation at 500 nanometers.

As shown in this graph, the oxidized beads show a significant emission peak at 538 nanometers compared to that of non oxidized beads with an intensity ratio of at least 11 to 12 fold. The generation of ROS in phagosomes in dium cells can be visualized qualitatively and dynamically by microscopy as shown in this representative time-lapse movie recorded for 40 minutes using six x magnification during the first minute after uptick. By phagocytosis, the fluorescence of OBG fluoresce coated beads changed from red to bright orange indicating that ROS were likely produced directly inside the phagosomes.

Intracellular superoxide and extracellular hydrogen peroxide production are quantitatively measured by the DHE and a UR assays respectively. A summary of the biochemical reactions related to these two assays are shown. Superoxide is converted to hydrogen peroxide by superoxide dismutase or SOD and hydrogen peroxide is converted to water and oxygen by catalase in the DHE assay.

A microplate reader is used for medium throughput quantitative measurement of intracellular superoxide. A representative experiment shows that when stimulated with LPSA lipopolysaccharide from e coli, the dynamic superoxide production from AX two cells is significantly higher than the basal level from non stimulated AX two cells and the background of the reaction in buffer, the decrease of blue fluorescence and the increase of red fluorescence are inversely correlated. As expected.

The localization of R os production measured by the DHE assay is confirmed by the following results. Addit addition of D-E-D-T-C-A membrane permeant superoxide dismutase inhibitor increased the DHE signal in a dose dependent manner indicating that D-E-D-T-C caused the accumulation of the superoxide dismutase substrate. Alternatively, addition of a membrane IMP hydrogen peroxide quencher did not affect superoxide production in the A UR assay.

A microplate reader is used for medium throughput quantitative measurement of extracellular hydrogen peroxide production. A representative experiment shows that when stimulated with LPS, the dynamic hydrogen peroxide production from AX two cells is significantly higher than the basal level from non stimulated AX two cells and the background reaction when treated with various concentrations of D-E-D-T-C or catalase, the A UR signal decreases in a dose dependent manner due to inhibition of hydrogen peroxide production from intracellular superoxide and depletion of extracellular hydrogen peroxide respectively. The treatments with D-E-D-T-C and catalase explicitly confirmed that the DHE and a UR assays specifically measure different types and subcellular localizations of ROS Indicum Following this procedure.

Other methods such as mirroring Ross generation during infection with pathogenic or non-pathogenic bacteria can be performed in order to answer additional questions such as how Dictal stallion will respond to different bacteria infection in terms of loss generation. And don't forget that working with pathogenic bacteria such as microbacteria marum can be extremely hazardous and precautions such as appropriate BSL two measures should be always taken while performing this procedure.