The hydrogen peroxide model is a simple and effective approach for evaluating the antioxidant effects of factors that may be useful in treating age-related macular degeneration or similar neurodegenerative diseases. The oxidant agent, hydrogen peroxide, is delivered in one single pulse, making it faster and simpler to perform than models in which the hydrogen peroxide treatment must be repeated for several days. Because the number of cells seeded influences the oxidative effect of the treatment, it is important to count the cells carefully before their culture.
Demonstrating the procedure will be Thais Bascuas, a post-doctorate from Gabriele Thumann's laboratory. For conditioned medium preparation, 28 days after transfection with PEDF, GM-CSF, or both, seed the human ARPE-19 cell line T-75 flasks out of 500, 000 cells per milliliter of complete medium concentration. When the cells reach approximately 80%confluency, replace the supernatant with fresh complete medium and collect the new supernatant after 24 hours.
To purify the histidine-tagged GM-CSF and PEDF proteins, centrifuge the supernatant to eliminate potential remaining cells, and set the sample aside. Next, add 30 microliters of nickel NTA solution to a 1.5 milliliter tube. After centrifuging, discard the nickel NTA flow-through and wash the pellet two times with 200 microliters of 1X incubation buffer per wash.
After the second wash, re-suspend the pellet in 40 microliters of 4X incubation buffer and 900 microliters of the centrifuged, conditioned medium. Incubate the conditioned medium solution for one hour at room temperature and 70 rotations per minute. At the end of the incubation, centrifuge the sample and wash the pellet two times with 175 microliters of 1X incubation per wash.
After the second wash, add 20 microliters of elution buffer for a 20-minute incubation at 70 revolutions per minute at room temperature. At the end of the incubation, centrifuge the sample. Set aside the supernatant and quantify the total protein eluted by the BCA protein assay according to standard protocols.
For the treatment of non-transfected retinal pigment epithelial cells with conditioned medium and hydrogen peroxide, seed 3000 cells in 200 microliters of complete medium to each well of a 96-well plate for 24-hour incubation in the cell culture incubator. The next morning replace the supernatants with 200 microliters of conditioned medium per well and return the cells to the cell culture incubators for 10 days. On day 11 of culture, treat the cells with 350-micromolar hydrogen peroxide in complete medium for 24 hours.
For the treatment of non-transfected RPE cells with purified or commercial PEDF and/or GM-CSF, seed 3000 cells in 200 microliters of complete medium supplemented with 500 nanograms per milliliter of recombinant PEDF and/or 50 nanograms per milliliter of recombinant to GM-CSF per well for a 48-hour incubation in the cell culture incubator. At the end of the incubation, replace the supernatants with complete medium containing 350 micromolar hydrogen peroxide and 500 nanograms per milliliter of PEDF and/or 50 nanograms per milliliter of GM-CSF per well for 24 hours. For hydrogen peroxide treatment of transfected RPE cells, culture 5, 000 transfected cells in 200 microliters of complete medium per well for 24 hours in the cell culture incubator before treating the cells with 350 micromolar hydrogen peroxide as demonstrated.
To measure the glutathione levels produced in transfected cells over-expressing PEDF and/or GM-CSF, or non-transfected cells pretreated with growth factor after hydrogen peroxide treatment, replace the supernatant in each well with 100 microliters of freshly-prepared 1X reagent mix per well and mix the cells with a reagent mix for 14 seconds at 500 revolutions per minute on an orbital shaker. After shaking, incubate the plate at room temperature for 30 minutes before adding 100 microliters of reconstituted luciferin detection reagent to each well. Mix the solution for 15 seconds on the shaker, followed by a 15-minute incubation at room temperature.
At the end of the incubation, load the plate onto a plate reader and select Change Layout. Under the Basic Parameters tab, 96-well plate and Top optic should be selected and the positioning delay should be set to 0.1, the Measurement start time to zero, the Measurement interval time to one, and the Time to normalize results to zero. Define the blanks, standards, and samples, and click Start measurement.
Then export the data as a spreadsheet file and calculate the concentration of glutathione in each sample by interpolation of the standard curve. To measure the cell viability, replace the supernatant in each well with 100 microliters of complete medium supplemented with 1%FBS per well, and place the cells in the cell culture incubator until the analysis. After collecting untreated ARPE-19 cells from culture, re-suspend the cells at a 100, 000 cells per milliliter in DMEM Hams F-12 medium supplemented with 1%FBS concentration.
Next, prepare seven serial 1:1 dilutions in 200 microliters of medium supplemented with 1%FBS, and transfer 100 microliters of each standard to the appropriate wells of a 96-well plate in duplicate. Add 50 microliters of freshly-prepared reagent mix to all of the well and mix the cells with the mix for 15 seconds on the shaker before incubating the plate for 15 minutes at room temperature. At the end of the incubation, measure the luminescence as demonstrated.
After obtaining the measurement, add 50 microliters of lysis reagent to each well for a 15-minute incubation at room temperature, then measure the luminescence again and use the formula to calculate the percentage of viable cells. 24 hours of treatment with 50-and 100-micromolar hydrogen peroxide does not impact intracellular glutathione production, whereas a significant decrease of glutathione is observed when the cells are treated with a 350-500-or 700-micromolar hydrogen peroxide concentration. Morphologically, hydrogen peroxide-treated cells appear less spread out and more rounded with increasing concentrations of the compound.
This effect was less prominent for PEDF and GM-CSF-transfected cells treated with hydrogen peroxide. It is important to note that cell number influences hydrogen peroxide-mediated oxidative stress, as the level of glutathione was statistically significantly decreased after hydrogen peroxide treatment in wells seeded with 5, 000 cells only. Cells treated with or transfected to express PEDF and/or GM-CSF produce significantly more glutathione compared to untreated control cells under oxidative conditions.
In addition, the level of UCP2 gene expression in PEDF and GM-CSF-transfected cells is increased after hydrogen peroxide, although the increase is not statistically significant. Western blot analysis of phosphorylated AKT from a lysate of GM-CSF-transfected cells exposed to hydrogen peroxide shows only a small decrease in phosphorylization compared to the untreated control cells, indicating that GM-CSF can protect the cells from oxidative stress damage. It is important to seed the cells at the correct densities, to use fresh hydrogen peroxide solution, and to add fresh proteins to the medium.
Once the antioxidant effect of the growth factors has been corroborated using the hydrogen peroxide model, the factors can be tested in an oxidative stress-related ocular disease model. The current hydrogen peroxide oxidative stress model allows assessment of the antioxidant effects that different factors can have, and therefore, supports the development of new gene therapies.
