Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

The overall goal of this procedure is to detect changes in cytosolic calcium in renal epithelial cells. This is accomplished by first loading the cells with a fluorescent calcium responsive dye in the presence of an anion transporter inhibitor probit. The second step is to bring the cells into suspension by gentle trypsin ization.

After centrifugation, the cells were washed in probit containing HBSS buffer. The final step is to measure compound induced changes using a flow cytometer. Ultimately, flow cytometry is used to show the kinetics of changes in cytosolic calcium in renal epithelial cells.

So main advantage of this technique over existing methods like lifestyle imaging, is that it is easy to perform quick and suitable for screening of compounds. This muscle can help answer key questions in the intracellular signaling field by allowing individuals to investigate the role of calcium in the context of physiological and pathophysiological cellular signaling. Demonstrating this procedure will be Sylvia Kyle, a technician from our group.

This experiment utilizes an immortalized adherent epithelial cell line derived from the S one segment of the rat kidney proximal tubule two days prior to calcium D loading plate, 2.5 times 10 to the five cells per well of a six well plate and standard culture medium. Grow the cells for two days to a co fluency of approximately 70%On the day of the experiment, prepare the calcium loading dye mixture for each well mix one milliliter of culture medium containing 5%fetal bovine serum. Four micromolar of the membrane permeable calcium binding dye flu oh 3:00 AM and two millimolar of probit.

Probit is an inhibitor of organic anion transporters and is used to prevent the efflux of calcium indicators. Replace the standard culture media in each well with one milliliter of the loading dye mixture. Cover the plate with aluminum foil to prevent bleaching of the fluorescent indicator and incubate for 30 minutes at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide it to prepare the cells for flow cytometry, aspirate the calcium loading dye solution from each well and add one milliliter of trypsin along the wall of the well.

Incubate at 37 degrees Celsius until all cells are detached. Transfer the cells to a 1.5 milliliter tube and pellet by centrifuging at 10, 000 times. G for one minute.

Carefully aspirate the supernatant without disturbing the pellet. After washing the cells with prevented containing HBSS flux or PHF buffer as detailed in the text protocol and resus suspend the cells in a final volume of 500 microliters of PHF buffer, the cells should be used for experimentation within one hour. Begin this procedure by starting the flow cytometer, opening the fluid extra and flipping the fent valve toggle switch.

To increase the air pressure of the sheath fluid tank, prime the instrument to remove any potential residing air bubbles from within the inner tubes and the flow chamber. In the flow cytometry software. Open two dot plot windows.

Set the flow cytometer to run. Then set flow rate to high. The flow cytometry measurements are performed at room temperature.

First at 480 microliters of PHF buffer to a flow cytometry sample tube. Then add 20 microliters of cell suspension and vortex briefly to mix. Move the tube support arm to the side and position the sample tube over the sample injection tube until a tight seal is formed.

Return the tube support arm to its original centered position. Optimize measurement by adjusting the fluorescence channel such that the mean fluorescence intensity of the sample is at approximately 10 to the second on the Y axis, save and use for subsequent experiments. To start an experiment, prepare another sample and position the sample tube over the sample injection tube.

As before, return the tube support arm to its original centered position. Record baseline fluorescence for 50 seconds. The next step must be performed quickly and accurately to ensure the signal is capture.

The success rate increases when everything is prepared. In each once Perform the following steps quickly in less than 15 seconds. Pause the measurement, remove the sample tube, add the compound of interest, vortex briefly, and return the sample tube to the sample injection tube.

Resume the measurement and continue for a total of 204.8 seconds. The data is analyzed offline using dedicated flow cytometry software. Open a dot plot with SSC and FSC parameters using the regions tool.

Select the region of cells for analysis to exclude dead cells or cell debris found in the bottom left corner. From the data analysis for a quadrant analysis, open a density plot with time on the x axis and fluorescence intensity on the Y axis, and use the same gating region as before. Quantify the data by using the quadrant tool.

Adjust the quadrant separators so that the vertical line is placed at the time of compound edition, and the horizontal line is placed at the center of the baseline fluorescence in the controls ensure that the positioning of the quadrant separators is equal in all samples. For a histogram analysis, open the control data in a histogram window with fluorescence intensity on the x axis and events on the Y axis in the non-treated control. Use the marker function to mark the area for analysis, starting from the midpoint between the minimum and maximum fluorescence of the control peak and ending at the maximum fluorescence intensity.

The percentage of cells in this area is then calculated for fluorescence intensity analysis. Open a dot plot with time on the x axis and fluorescence intensity on the Y axis. Quantify the relative calcium influx by creating multiple rectangular regions.

Each region should have a width of about 50, which is equivalent to 10 seconds. Define up to 15 rectangular regions. Use the why mean data for analysis.

Copy data into a spreadsheet table application calculate the mean of R two to R five, which is equivalent to 20 to 50 seconds. This is the baseline value. After choosing an appropriate time interval for calcium influx analysis, EEG R seven to R 11 calculate the calcium signals from each region within this time interval relative to the mean baseline value that is the fluorescent signal before compound addition, which is set to 100%In this protocol, the integrity of the cell population is assessed using side scatter and forward scatter.

A region is selected for further analysis to exclude dead cells and cell debris that have reduced light scattering. A density plot for fluorescence intensity versus time was created for each data file. The cells were treated with FAFSA gargen and tcom mycin Iono Mycin was used as a positive control.

Each dot plot was separated into four areas representing fluorescence intensity before and after compound addition and above and below the baseline fluorescence. As an example, the percentage of cells in the upper right quadrant increased from 42.8%in the untreated control to 51.3%in thic argen treated cells, indicating that a calcium signal was induced. A histogram analysis showed fluorescence intensity increases of 1.41 fold, 1.36 fold and 2.11 fold by FAFSA gargen, tcom mycin, and iono mycin respectively.

The multiple region analysis mode revealed that thsa, gargen, T Mycin and Iono mycin increased the calcium signal by 1.55 fold, 1.29 fold and 4.54 fold respectively to determine whether transient physiological calcium signals can be measured. A TP was applied to the cells. Data was quantified using multiple rectangular region analysis, quadrant analysis or histogram analysis.

A TP evoked a rapid and transient calcium signal, which was only detected by the multiple rectangular region analysis mode with selected parts of the trace The following, this procedure, other methods like lifestyle imaging can be performed in order to gain further information about the calcium signals such as spatial changes in calcium and the immediate kinetics of the calcium signal. After watching this video, you should have a good understanding of how to detect relative changes in cytosolic calcium in the cells using a flow cytometer.