The overall goal of this procedure is to determine tolerable fatty acid and cholera toin concentrations that do not negatively influence cell survival. This is accomplished by first growing the appropriate type of cells. The second step is to incubate the cells with varying concentrations of the three fatty acids to be tested or with the cholera toxin.
After 24 hours, cell survival is assessed using an MTT assay. Ultimately, spectrometry data from the MTT assay show optimal concentrations of fatty acids and cholera toxin with which cells can be treated without significant reduction in survival. The implications of this technique extend towards the prevention of cholera infections because vaccinations are not always successful.
Moose musculus macrophages from bowel C mice will be used for all co toxin determinations and for larger scale concentration determination of fatty acids, the cells are propagated in either of two media, DMEM with L glutamine completed with 10%fetal bovine serum and 1%antibiotic antimycotic or RPMI 1640 base media completed with 10%fetal bovine serum, 5%L-glutamine, and 1%antibiotic. Antimycotic for this protocol, grow the bowel C cells in 75 centimeter square Corning flasks with vented caps at 37 degrees Celsius, 95%air and 5%carbon dioxide for fine scale. Fatty acid concentration determination use human intestinal epithelial cells propagated in 75 centimeter square Corning flasks with vented caps in hypercare media completed with 10%FPS and 30 nanograms per milliliter.
Human EGF do not use antibiotic antimycotic reagents as per manufacturer's instructions incubate at 37 degrees Celsius, 95%air and 5%carbon dioxide subculture, all cells one to three at approximately 70%Co fluency, depending on the type of cell cells, are typically subculture every two to five days prior to the fatty acid treatment. Prepare the fatty acids first transfer oleic, linoleic, and linoleic acids from the company provided glass ampules to sterilized glass vials. Next, transfer each fatty acid to a separate sterilized einor tube.
Dissolve each fatty acid in 100%Ethanol at a dilution of one to six, followed by edition of RPMI 1640. Incomplete media to a final concentration of 10 micrograms per microliter. Vortex each solution and transfer them to glass vials for storage in the minus 20 degrees Celsius freezer plate.
Human intestinal epithelial cells at 2, 500 cells per well in 96. Well tissue culture plates add the appropriate complete media to bring the total volume of each well to 200 microliters. Incubate the cells at 37 degrees Celsius, 95%air and 5%carbon dioxide for 24 hours.
After the 24 hour proliferation period, remove the media. Add the appropriate concentrations of each fatty acid to be tested to the wells as a positive control. Incubate cells with complete medium as a negative control, incubate cells with 70%ethanol.
After that, add complete media to bring the total volume of each well to 200 microliters. Incubate the treated plates for 24 hours before beginning the MTT assay to prepare the cholera toxin for cell treatments. Dissolve the cholera toxin in PBS at a concentration of one milligram per milliliter.
Aliquot the solution in cryo vials and store at two to eight degrees Celsius prior to cell treatments. Dilute the toxin one to a hundred in either sterilized PBS or incomplete DMEM for a final working concentration of one nanogram per microliter plate. Macrophages at 2, 500 cells per well in 96.
Well tissue culture plates incubate at 37 degrees Celsius, 95%air and 5%carbon dioxide for 24 hours. After the 24 hour proliferation period, remove the media and add the appropriate test concentrations of cholera toin to the wells. Similarly to the fatty acid treatments, incubate cells with complete medium as a positive control and 70%ethanol as a negative control for all samples and treatments.
Use a minimum of three replications with more replications for positive controls. Add complete media to bring the total volume of each well to 200 microliters. Incubate the cholera toxin treated plates for 24 hours before beginning the MTT assay.
24 hours after cells have been treated with either fatty acids or color toin cell viability is determined by the MTT assay. Remove the solution in each well of the 96 well plate to be tested and replace it with 200 microliters. A fresh complete media solution.
Add 10 microliters of a freshly prepared 2.5 milligram per milliliter MTT solution to each. Well incubate the plate at 37 degrees Celsius for three to four hours. After three to four hours, discard the media solution and add 100 microliters of 0.04 molar hydrochloric acid in isopropanol to each.
Well incubate the plate at room temperature for five minutes. Next, transfer the solution from each well to a centrifuge tube centrifuge at 20, 000 GS for one minute or until a pellet is formed. Transfer between 20 and 40 microliters of each sample to a microplate reader and read the absorbance at 570 nanometers using a spectrophotometer.
This figure shows the survival of most macrophages depicted as a function of the positive control for treatments using increasing concentrations of oleic acid, linoleic acid, and linoleic acids. It is evident that the cells do not tolerate concentrations above 100 nanograms per microliter for any of the fatty acids. The experiment was repeated using human epithelial cells for concentrations that were lower than 100 nanograms per microliter in 10 nanogram per microliter increments, up to 70 nanograms per microliter.
This graph shows cell survival for each concentration of oleic acid. One way Inova indicated that one or more of the means were statistically different. However, it is only the treatments at higher concentrations that rendered statistically different means from that of the positive control.
Interestingly, the differences observed were due to an increase in cell viability with more variability. Similar results were obtained for linoleic and linoleic acids. One way.
Inova indicated that none of the treatment means were statistically different from the mean of the positive control, but variability seems to increase for some of the higher concentrations, such as 60 nanograms per microliter of linoleic acid or linoleic acid from these results. The optimum fatty acid concentrations when using human intestinal epithelial cells appear to be between one and five nanograms per microliter. The effects of varying concentrations of collar toxin on most macrophage survival were also tested with treatments for toxin levels at relatively large increments.
A decreasing viability trend for cell survival was observed as expected cell viability varied greatly for each treatment. The large variability in viability resulted in no significant difference between the mean of the positive control with any of the treatments, which led to further testing of toxin levels of 100 nanograms or lower. This graph shows cell viability for the smaller toxin level treatments.
Cell viability is highly variable for almost all toxin concentrations, albeit slight increases were observed in viability as a result of the treatments. These increases are not statistically significant for any of the treatments compared to the mean of the positive control. Based on these results.
The recommended concentration of color toxin is less than 30 nanograms per treatment when using bowel C mouse macrophages After its development. This technique paved the way for researchers in the field of color or research to explore whether fatty acids may enhance immunity against infection by vio cholera.
