The overall goal of this procedure is to use an impedance based technique to follow the real time cyto protection of a neuronal cell line to delineate the involvement of second messenger pathways in the protection process. This is accomplished by first culturing a neuronal cell line under proliferative conditions in an impedance based realtime cell analyzer. In the second step, potentially relevant.
Second messenger pathways are pharmacologically inhibited before subjecting the cell line to neuroprotective treatments. Next, the status of the neuronal cell line is followed using a real-time cell analyzer under the different experimental conditions. Ultimately, statistical analysis can be used to assess the real-time involvement of second messenger pathways in the cyto protection of the neuronal cells.
The main advantage of this technique over existing methods like endpoint neurotoxicity assays, is that it allows Tito protection and tito toxicity to be evaluated in neuronal cell lines in real time and under label free conditions. This Method can help answer quick questions in the molecular neurobiology field by contributing to more high throughput assessment of potential neuroprotective compounds in vitro, and by providing insight into the molecular mechanisms involved in neuroprotection Begin by washing an adherent neuronal cell line layer. For example here, human neuroblastoma S-K-N-S-H cells are used with PBS then trypsin the cells for five minutes at 37 degrees Celsius.
Remove the trypsin by centrifugation and then resuspend the cells in 10 milliliters of proliferation medium. After counting the cells dilute the cells to a three times 10 to the fifth cells per milliliter concentration with more proliferation medium, then equilibrate each well of an EPL 96 with 100 microliters of proliferation medium in a tissue culture hood at room temperature. After 30 minutes, insert the EPL 96 into a real-time cell analyzer station in a carbon dioxide incubator at 37 degrees Celsius and start the real-time cell analyzer software.
Then on the layout page, select the appropriate wells for the experiment and enter the cell type cell number and the names and concentrations of the chemical compounds of interest into the edit boxes on the schedule software page as step one is preset. For the background measurement, select add a step and set step two to measure the cell index every 15 minutes for 96 hours. Then click start to initiate step one, and to measure the background impedance of the media.
Now remove the EPL and add 100 microliters of the neuroblastoma cell suspension. To each experimental well gently swirl the EPL 96 to evenly coat the wells, and then incubate the plate at room temperature in the tissue culture hood to allow the cells to settle after half an hour, load the EPL 96 back into the real time cell analyzer station and start step two. To monitor the cell index values throughout the experiment, view the cell curves on the plot page and the raw cell index data on the cell index page 24 hours.
After plating the cells, pause the experiment and remove the plate from the realtime cell analyzer station. Dispense one microliter of the freshly diluted inhibitors of interest into each well then after reloading the EPL 96 resume the experimental cell index measurements after 30 minutes, pause the experiment again and quickly but carefully remove the proliferation medium from each well directing the edge of the pipette tip to the corner of the well to avoid disrupting the adherent cell layer. Then immediately add 200 microliters of serum deprivation medium to each well, one microliter of the freshly diluted neuroprotective compounds of interest, and one microliter of the second messenger pathway inhibitors of interest to the appropriate wells.
Then continue with the cell index measurements every 15 minutes for 96 hours as previously set to analyze the data, now select the normalized cell index and normalized time functions on the plot page to normalize the cell index to the last time point before the modifications to the cell culture. To reduce the variation between the experiments. Highlight the wells on the plot page for the experimental conditions of interest and click add to plot the normalized cell index curves.
Then export the experimental data for all the cell index time points from the cell index software page into a spreadsheet file for statistical analysis of the differences in the cell index values under the different treatment conditions at specific time points. Select the cell index values at their respective time points within the exported spreadsheet file. Finally, analyze the cell index values for the selected time points with one or two way analysis of variance, followed by a post hoc test Using statistical software neurotoxic stimuli to the cells lead to a decrease in the cell index values here an increase in the cell index when the S-K-N-S-H cells are grown in proliferation medium and the drop of the cell index followed by a stabilization at the new lower levels after switching the cells to serum deprivation medium are shown in these graphs.
The neuroprotective effect of two serotonin two a receptor agonists five micromolar, DOI and 20 micromolar IDE added when the cells were fed with serum deprivation, medium can be observed. An important question in neuroprotection studies is whether an effect on the cell proliferation contributes to the observed compound effect on the cell survival. In this graph, the lack of effect of five micromolar DOI treatment on SKNS cell proliferation is shown suggesting that the proliferation does not contribute to the observed neuroprotective effect.
In this final graph, the effect of treatment with 10 micromolar of the phosphatidyl inital three kinase inhibitor LY 2 9 4 0 0 2 on cell survival and the neuroprotective effect of DOI on serum deprived SK NSH cells can be observed. LY 2 9 4 0 0 2 prevented the neuroprotective effect of DOI during the serum deprivation stage, but also exhibited some toxic effect of its own. After watching this video, you should have a good understanding of how to follow CTO protection in a neuronal cell line using a real time cell analyzer, and to delineate the involvement of second messenger pathways in protection.
