The overall goal of these multi-well based sequence assays, is to better understand the effects of cold physical plasma over oxidants, to biological responses in vitro, Using optical emissions spectroscopy, liquid analysis, and cellular activity, microscopy, and flow cytometry assays. This method can help to answer key questions in the field of plasma medicine, especially with regard to plasma derived directive species, important for the induction of immunogenic cancer cell death. The main advantage of this technique is it's semi-automatized approach.
It uses 96 well plates, to increase speed and productivity of the research. The implications of this technique, extends out to future plasma therapies, like in dermatology or oncology. And, as they provide the means to identify the plasma derived directive species, which are relevant for the biological responses.
Demonstrating the procedure will be Felix Niessner, a technician from our laboratory. For plasma species monitoring, position an atmospheric plasma jet in front of an optical emissions spectrophotometer, perpendicular to the plume axis, and use the dedicated software to record the photoemission and wavelength of each gas condition, at two standard liters per minute feed gas flux. For plasma treated super oxide analysis, set up a final protocol for the XYZ table, with the appropriate respective treatment times and well positions.
Then, add 100 microliters of freshly prepared master mix to the wells of a clear, flat-bottomed 96 well plate in triplicate, and use a micro plate reader, to measure the absorbance at 550 nanometers. To analyze the effects of plasma treatment on cell metabolic responses, in a laminar flow hood, seed one times 10 to the four cells of interest in 100 microliters of fully supplemented cell culture medium, per well, in a flat-bottom 96 well plate, and incubate overnight. After overnight culture in a cell culture incubator, use the XYZ table to treat the wells with plasma or gas alone, according to the experimental analysis, and return the cells to the incubator for another 20 hours.
At the end of the second incubation, add 25 microliters of fresh cell culture medium, supplemented with 500 micromolar Resazurin to the cells, as well as to the three background control Resazurin-only wells. Return the cells to the incubator, for a three hour incubation. Then, measure the fluorescence in a micro plate reader.
For imaging of the plasma-treated cells, after reading the fluorescence, replace the supernatants with 100 microliters of fresh cell culture medium, supplemented with one microgram per milliliter of propidium iodide. Place the plate on a motorized microscope stage, And select the 20x objective to image the cells. Then, use the appropriate quantitative image analysis software to determine the total cytosolic area in digital phase contrast images, of all of the fields of view imaged in each well.
For flow cytometric analysis of the plasma treated cells, after imaging, wash the cells in each well two times with 200 microliters of PBS, supplemented with calcium and magnesium per wash, followed by labeling, with 15 anagrams per milliliter of anti-mouse calreticulin monoclonal antibody, in 50 microliters of PBS, plus calcium and magnesium per well. After 15 minutes in the cell culture incubator, wash the cells two times with 200 microliters of complete cell culture medium, and add 100 microliters of cell detachment solution to each well for 20 minutes, at 37 degrees Celsius. When the cells have detached, load the plate onto the flow cytometer, and acquire a minimum of 1, 000 events in the forward and side scatter population, usually associated with viable cells.
Then, use the appropriate flow cytometry analysis software to gate the population of interest, and to determine the mean fluorescence intensity of the calreticulin expression. Optical emissions spectroscopy can be used to follow the distinct peaks linked to reactive plasma components in different feed gas conditions. For the plasma treatment of liquids, the evaporation caused by the argon gas and the argon plasma is determined first, with the conditions yielding different evaporation results, as plasma also exerts effects on temperature.
Similar to the optical emissions spectroscopy results for hydroxyl radicals, hydrogen peroxide deposition significantly decreases with oxygen or nitrogen admixture, but increases with humidified feed gas. Moreover, the addition of nitrogen to the feed gas leads to significantly higher nitrate concentrations, compared to argon plasma treated liquids. Most super oxide is produced under dry argon gas conditions, with oxygen and or nitrogen admixtures significantly quenching the super oxide generation, except in the presence of humidified argon oxygen plasma.
The fluorescence intensities of Resazurin and plasma treated cells are similar to the visually observed physical changes to the supernatants of the cell cultures, confirming the cytotoxic effects of prolonged plasma treatment. A decrease in the total cell area is also observed in the plasma treatment sample wells, particularly under the humidified feed gas conditions, with an overall up regulation of calreticulin staining on miren melanoma cells, in response to shorter exposures to plasma treatment. If performed properly, the mytoplex cellular assays and the readout can be completed in only a few hours.
Following this procedure, other methods can be performed as well, such as the co-culture with immune cells, or the analysis of cell culture supernatants. This helps to answer additional questions, about the release of dams, cyto clients, or redux proteins, as well as the immune recognition of plasma-treated melanoma cells. After it's development, this technique paved the way for further research into melanoma cycles, for instance with 3D tumor spheroids.
After watching this video, you should have a good understanding of how to perform basic research in plasma medicine, from the plasma gas phase, over reactive molecules and liquids, to biological responses in melanoma cells.
