Hello everyone, my name is Dr.Bipasha Bose. I work as an associate professor and in charge in the Stem Cells and Regenerative Medicine Center, Yenepoya Research Center, Yenepoya University, Mangalore, India. Now we are going to demonstrate how to detect for the presence for ROS that is, reactive oxygen species"in the live cultures from the mouse ocular surfaces that have been exposed to various doses of ultraviolet-C radiation.
The advantage of this technique is that here we can simultaneously detect for the presence of reactive oxygen species, live and dead cells, without the need to have Vester cells. The principle of this technique essentially lies in the live cell permeability of the dye DCFDA. DCFDA is a live cell permeant colorless dye, which during oxidative stress gets acted upon by the intracellular oxidases and gets converted into green fluorescing DCF.
Hence, the green florescence enables us to detect for the presence of reactive oxygen species in the live cells. On the other hand, propidium iodide is an exclusively dead cell permeant dye which fluoresces red. It is a rDNA double-stranded intercalating dye.
However, the dye Hoechst is permeant to both the live and dead cells. It is a blue color nuclear stain. Hence, this is a very simple method in which we can detect for the presence of reactive oxygen species in long-term cultures in a time dependent manner along with the assessment of dead cells.
Plate point two million cells per 35 millimeter culture dishes. The cells we're applying are the cells from mouse ocular surface. Remove maximum volume of media from each of the cell culture dishes.
Leave behind around 500 microliters of cell culture media in close contact with the cells. Minimal amount of media will prevent the drying of the cells. However, the purpose of minimal amount of media is to allow the maximum penetration of UVC exposure.
Take the cells under a UV source, it can be either a UV Crosslinker, or can be any other ultraviolet-C source. Expose the cells to different dosage of UVC radiation such as one, 10, 100, 1, 000, and 10, 000 joules per meter square. When the cells have exposed to ultraviolet-C radiation, the lids should be in the open position.
This will allow maximum penetration of the ultraviolet-C to the cells and hence show the optimal dose-response of the cells to the ultraviolet-C radiation. Bring the cells to the laminar air flow hood and replenish each of the dishes with two milliliter of complete media. This complete media contains 20%fetal bovine serum in DMEM, supplemented with supplements such as 1%minimum non-essential amino acids, and also 1%antibiotic that is penicillin and streptomycin.
Afterwards, after replenishing with maximum volume, that is two milliliters of complete media, return the plates to the incubator and incubate for a period of three hours in order to see early effects of ultraviolet-C radiation. Prepare the live cell staining media in the last 15 minutes of three hour post UVC cell incubation. Staining media is prepared in 10%FBS containing DMEM pre-warmed to 37 degrees.
For making 10 milliliter of staining media, first add five microliter of DCFDA from a stock of 10 millimolar so as to obtain a final concentration of five micromolar. Mix well by pipetting up and down. Secondly, add five microliter of Hoechst solution from a stock of 10 milligram per mL to obtain a final concentration of five microgram per mL.
Now mix well by pipetting up and down. Lastly add 200 microliters of propidium iodide from a stock of one milligram per mL to obtain a final concentration of 20 micrograms per mL. Mix well by pipetting up and down.
Now the staining solution is ready for use. After three hours of incubation post UVC exposure, remove the plates from the CO2 incubator. Aspirate out the media from each of the dishes which have been exposed to various dosage of UVC such as one, 10, 100, 1, 000, and 10, 000 joules per meter square.
Now add two milliliter of freshly prepared live cell staining media to each of the dishes gently from the sides of the dishes. Return the plates to the CO2 incubator again and incubate for 15 minutes for live cell staining. After 15 minutes of incubation in the live cell staining media, remove the staining media from each of the dishes containing cells exposed to different doses of ultraviolet-C radiation.
Replenish the cells with two milliliter of complete media. Now the cells are ready are ready for viewing. Now place the control cells under the bright-field of the fluorescent imager.
The cells look apparently normal since they are control cells. Under the blue field we can the fluorescing total cells. Under the green field shows the ROS generation.
Since these are control cells, there is no ROS generated. Under the red field, the propidium iodide stained dead cells are visible. Then keep the UV exposed 100 joules per meter square under the bright-field.
Under the blue field, and also we can see the total cell number under the blue field. However, when we expose to green field, DCFDA positive cells are seen, and also PI positive dead cells are seen. Finally, place the maximum UV dose, that is 10, 000 joules per meter square exposed cells, under the bright-field.
We can see abnormal cell morphology. However, under the blue field we can see total blue cells. Under the green field, green fluorescing DCFDA positive cells are seen indicating the ROS generation.
When the cells are exposed to red field, all the cells were fluorescing red indicating a large number of cell death when the cells are treated with 10, 000 joules per meter square of UV dosage. Now arrange the images captured in various channels into a single composite image panel corresponding to the ultraviolet-C doses and unexposed controls. The images are arranged in a single panel in group.
First, the bright-field. Second, the Hoechst blue nuclear stain. Third, propidium iodide staining as for dead nuclei.
Fourth, DCFDA for ROS positive cells. And fifth, the merged image. When we look at the first and the second row of images, that is unexposed control and the cells exposed to the UVC dose one joule per meter square, we observed that there was neither PI nor ROS positive cells that had lighted up, thereby indicating a complete absence of ROS and cell death in unexposed controls and such a low UVC dose of one joule per meter square.
Now coming to the third row of composite images of the cells that were exposed to 100 joules per meter square. A very low percentage of cells, about 10%were positive for both PI and DCFDA at this dose, thereby indicating low amount of ROS generation and cell death. Now we move on to a further higher dose of UVC radiation exposure, that is 1, 000 joules per meter square as indicated in the fourth row of the composite image.
Here, about 70%of the cells were positive for PI and DCFDA. Finally, we move on to highest dose of UVC exposure, that is 10, 000 joules per meter square as represented in the fifth row of the composite image. Here were find that almost 100%of the cells were positive for both PI and DCFDA, thereby indicating a 100%cell death and ROS generation at this particular UVC dose.
Transfer the images to the imaging software. First open the blue image indicating the Hoescht stained nuclei, that is the total number of cells. Open the counting tool and click each of cells one at a time in order to have the number.
Now open the image captured under the red channel indicating the PI positive dead cells. Open the counting tool and click each of the red spots indicating the count for the PI positive dead cells. Once the counting of the dead cells is over, click on the green channel captured cells and open the counting tool and click each of the green spots indicating the cells showing the ROS generation.
Complete the counting of the green cells captured under the green channel, and then using the formula enumerate the percentage of cell death by UVC damage and percentage of ROS production by UVC damage. This is calculated using the simple formula number of PI positive cells, that is the red fluorescing cells, divided by number of Hoechst positive cells multiplied by 100. Percentage of ROS production by UV damage is calculated using the formula, number of DCFDA positive, or green fluorescing cells, divided by number of Hoechst positive cells multiplied by 100.
Once you have both the percentages, that is percentage of cell death and the percentage of ROS production, use these values to plot a bar graph. X-axis indicates the dosage of UV, whereas y-axis indicates the percentage of cells. The green bars indicate the percentage of ROS generation, whereas the red bar indicate the percentage of cell death.
Upon analysis, it is evident that at UVC dose 100 joules there is 10%ROS generating cell as well as a 10%cell death. Whereas at the UVC dose 10 raised to three joules per meter square, 70%cells exhibited ROS generation as well as cell death. While at the highest dose of UVC, that is 10 raised to four joules per meter square, about 100%cells exhibited cell death as well as ROS production.
Hence, it can be concluded that there is a strong positive correlation between the ROS generation and cell death. In conclusion, this technique is very handy for simultaneous assessment of reactive oxygen species, live and dead cells in live, normal event culture. This technique is also useful for limited assessment of reactive oxygen species, live and dead cells, in long-term cultures which have been exposed to various cell damaging agents such ultraviolet radiation, or chemical agents.
And this technique can also guide a researcher for determining the optimum time for harvesting the cells such at 50%ROS, 75%ROS, so and and so forth as there can be for many downstream applications, such as QR to PCR and Western blotting.
