The overall goal of this video is to demonstrate a high output multiplex assay to determine the onset of apoptosis as measured by caspase. Three seven. First hypothalamic neurons are challenged with the apoptotic inducing saturated fatty acid, palmitic acid.
Then the cell viability of the neurons is measured using a fluorescent based reagent and a microplate reader following the onset of apoptosis. The lumino substrate, DEVD, which is an amino acid sequence cleaved by caspase three, is added to the cells. The final step of the procedure is to measure luminescence using a microplate reader to determine caspase activity, which increases in cells that are undergoing apoptosis.
Ultimately, a ratio of caspase activity per viable cells can be obtained in less than five to six hours. In a 96 well format. The main advantage of this technique of our existing methods, like western blots or lysis, is that is a rapid, inexpensive assay that can directly measure caspase three seven activity in multiple well formats.
This method can help answer key questions in the field, such as determining primary modulators of apoptosis. The utility of this technique is that this multiplexing can be used as an accurate method of assessing cytotoxicity and viability. Although the assay is fairly straightforward, a visual demonstration of this method is critical because it will ensure that the assay will be successfully completed.
Though this method can provide insight into neurodegeneration, it can also be applied to other diseases and in vitro models such as those used in cancer research. Individuals new to this method will need to optimize this technique because the endpoints are cell line and agonist dependent. Caspase three seven activity is transient.
Therefore, depending on the effectiveness of the cell stressor, determining the appropriate timing to assay cast base activity can take some effort To begin rapidly. Thaw frozen a 12 cells in a 37 degrees Celsius water bath. Once thawed, gently pipee the cells into a 75 centimeter squared vented culture flask and add 10 milliliters of warm delcos, modified eagles, medium, or DMEM, incubate the flask overnight in a 5%carbon dioxide incubator at 37 degrees Celsius.
After 24 hours, aspirate the medium and replace with fresh medium can continue growing cells until between 70 and 80%Confluence is obtained after two to three days of growth. To count the cells first warm DMEM and one x trypsin, EDTA solution to 37 degrees Celsius. Aspirate the media from the flask and wash two times with sterile phosphate buffered saline.
Add 500 microliters of tryin solution before incubating at 37 degrees Celsius for between two and five minutes. Detach the cells from the flask using a scraper and suspend cells in five milliliters of media. Pass the cells through a cell strainer into a clean 50 milliliter tube.
It may be necessary to pass the cells through the strainer more than once, but do not repeat more than three times after counting the cells using a hemo cytometer seed. 6, 000 cells per well in a 96 well clear bottom black or white walled plate in a final volume of 100 microliters of medium, incubate the plate at 5%carbon dioxide and 37 degrees Celsius for 24 hours. Transfer palmitic acid to a sterile five milliliter glass vial and solubilize in 100%DMSO.
After diluting the stock palmitic acid solution one to 10 in DMSO, add it to PREWARM DMEM to obtain a final working concentration of 0.1 millimolar palmitic acid for control media, add the same concentration of DMSO that was added to media containing palmitic acid. Remove the media in each well and replace with 50 microliters of medium plus or minus tic acid, and incubate the plate at 5%carbon dioxide and 37 degrees Celsius for two hours. Then add five microliters resin reagents, and incubate for 10 minutes at room temperature following incubation, load the plate onto a multimode Microplate reader and record fluorescence at an excitation wavelength of 560 nanometers and an emission wavelength of 590 nanometers.
To measure cell liability results are presented as relative fluorescence units to the same plate. Add 55 microliters of DEVD cast reagent and incubate at room temperature for two hours. Again using the Microplate reader record luminescence to measure caspase three seven activity results are presented as relative luminescence units with luminescence directly proportional to caspase three seven to normalize caspase three seven activity to cell count, divide cell viability by caspase three seven activity.
This protocol describes results in multiplexing two separate assays to determine mechanisms of cell death by apoptosis. Caspase activity was significantly increased in cells challenged with palmitic acid. After two hour incubation, cell membrane integrity is visibly altered in the presence of palmitic acid.
Shown here is the potential pathway in which palmitic acid induces apoptosis. Understanding the underlying mechanisms of the stressor used to induce apoptosis aids in determining the length of time for the incubation of stressor. Since caspase enzymes must be activated before interacting with the DEVD substrate Once mastered, this technique can be done in less than five to six hours if it is performed properly.
After watching this video, you should have a good understanding of how to perform a multiplex caspase three seven activity assay using an in vitro hypothalamic neuronal cell culture model. While attempting this procedure, it's important to remember that incubation times are dependent on cell types and stressors or agonists used. The development of this technique allows researchers in the field of neuroscience to use a reliable and time-saving technique for apoptotic studies Following this procedure, additional pharmacological assays that target specific proteins within the apoptotic process can be performed in order to delineate underlying mechanisms that initiate degenerative diseases.
