Our methods outline a simple and repeat way for studying nanotoxicity, such as examining the types of cell death and examining the production of reactive oxygen species. Fluorescence activated cell sorting is a strict format that allows us to study the subpopulation of cells and the growing different types of cell death. The DHE probe study method allows us to detect and quantify reactive oxygen species followed by flow cytometry, microscopic high-content imaging, and by couplet based fluorescence analysis.
To begin, place an Eppendorf tube containing zinc oxide nanoparticles in zinc oxide nanoparticle stock solution into a sonicator for 15 minutes. Prepare zinc oxide nanoparticles at various concentrations using one-milligram-per-milliliter zinc oxide nanoparticle stock. Then, in a biosafety cabinet, seed MRC5 human lung fibroblasts onto three six-well culture plates at the concentration of one times 10 to the fifth cells per well.
Incubate the cells at 37 degrees Celsius. After one day, treat the cells with two milliliters of zinc oxide nanoparticles for eight hours, 16 hours, or 24 hours. At each time point, collect the cells into a 15-milliliter centrifuge tube, and centrifuge at 300 times g for five minutes.
Wash the cell pellets twice with phosphate-buffered saline, and then resuspend the cells with 1X binding buffer. Next, add five microliters of FITC Annexin V stain in five microliters of propidium iodide DNA stain. Incubate the stained cells for 15 minutes at room temperature in the dark.
Before sorting the cells by flow cytometry, top up the samples with an additional 400 microliters of 1X binding buffer. Tabulate the bar chart using the median intensity obtained. Now use a pipette to add one milliliter of nanoparticles at different concentrations into vials, followed by nine milliliters of fly food to make a final concentration of 0.1 milligrams per milliliter, 0.25 milligrams per milliliter, or 0.5 milligrams per milliliter zinc oxide nanoparticles.
Pipette up and down to mix thoroughly. Allow fly food containing zinc oxide nanoparticles to cool for at least two to three hours before use. Then anesthetize adult male and female flies in a vial with carbon dioxide, and introduce five male flies and five female flies into each vial for five days.
Allow them to mate and lay eggs on the surface of the food. After anesthetization, remove the parental flies and allow the eggs to undergo further development at room temperature, which consists of four different developmental stages. Embryonic, larval, pupal, and adult stage.
After 72 to 120 hours, freshly-laid eggs develop into late third instar larvae. Using forceps, collect the larvae from the wall of the vials for analyses. In a well of a dissection dish with dissection medium or PBS, place the larvae into the solution.
Under the stereomicroscope, use the tip of the forceps to make a tiny hole, and break open the cuticle layer of the larvae. Carefully pull out the gut. Place the gut into a 1.5-milliliter microcentrifuge tube containing Schneider's Drosophila Medium.
Add one microliter of the DHE probe, and incubate in the dark at room temperature for 10 minutes. Wash the gut three times using Schneider's Medium every five minutes. Mount the gut onto glass slides, and supply with 10 microliters of antifade mounting medium containing DAPI.
Capture images under a confocal microscope. To measure fluorescence, import the captured fluorescence images acquired using fluorescence microscopy or confocal laser scanning microscopy into the ImageJ software. Click on the analyze menu, and select set measurements.
Select the output measure, such as area integrated intensity and mean gray value. Select a region without fluorescence to set the background. Click measure.
Export the data into the spreadsheet, and determine the corrected total cell fluorescence. Construct a bar chart, and perform statistical analysis. In this study, zinc oxide nanoparticle-treated cells exhibit a higher percentage of early R3 or late apoptotic R6 cells than control cells R5.Necrotic cell death was denoted by R4.The gut extracted from the drosophila larva was divided into three discrete domains of different developmental origin.
Namely, the foregut, midgut, and hindgut. DHE stock tends to expire rather quickly, so be careful of the stock solution you are using. Alternatively, you could try other probes, for example cell Roche, which is designed to detect the intracellular ROS in green signals, and that is more photostable than the DHE.
Our protocol provides a general outline for studying the intracellular ROS production, and quantification of ROS level for nanotoxicity study. DMSO is known to be a better solvent for DHE than the PBS. However, DMSO is toxic.
Therefore, I would like to advise the user to restrict the entire process for studying to 30 minutes maximum. This is because you may observe a false positive outcome as DMSO may cause cell death.
