The overall goal of this procedure is to dissect rapid calcium signals of different durations. This is accomplished by first culturing cells on a slide or well that allows for cell aian and imaging. Next, the cells are loaded with a fluorescent calcium indicator.
Then the cells are micro injected with a drug or other protein of interest. The final step is to carry out rapid fluorescence imaging on a confocal microscope. Ultimately, the calcium responses of different timescales can be evaluated.
My name is ULA Ska. I am a research associate in Dr.Scar laboratory. This method can help answer key questions in the field of cell signaling, such as how a particular drug can alter calcium mobilizing pathways in cells.
The implication of this technique extend toward the therapies of cardiac arrhythmias. Since cardiac arrhythmias are told to be due to disruption in basal calcium currents The day before beginning the procedure coat 35 millimeter glass bottom viewing chambers with 10 micrograms per milliliter of laminin. The next day plate, one milliliter of freshly isolated cells suspended in KB buffer in the chambers for 45 minutes after the cells have endeared to the bottom of the chambers, gradually change the buffer to M1 99 medium supplemented with FBS, streptomycin and Gentamycin, and then incubate the chambers at 37 degrees Celsius in 5%carbon dioxide for 24 hours.
After the incubation, stain the cells with the desired calcium indicator for 30 to 45 minutes at room temperature, covering the dish with aluminum foil to avoid photo bleaching of the dye. Finally wash the cells three times with the appropriate viewing media and then incubate the cells for another 30 to 45 minutes of room temperature in lie of it's 15 medium supplemented with EGTA. After culturing the cells in the glass bottom, viewing chambers for 24 hours.
Change the medium to phenol freely of its 15 with EGTA. Then load an automated microinjection system with a reasonably concentrated D solution, such as the CAV three scaffold protein and DPI solution used here. Next, set the injection and compensation pressures of the Microinjection system to 90 Hector Pascal and 45 Hector Pascal respectively and set the injection time T to 0.7 seconds.
Readjusting the injection parameters as needed. Performing micro micro injection is one of the most difficult parts of this procedure. While administering the dice solution, one has to remember to minimalize the damage to the cells.
Finally, using an inverted microscope equipped with a long working distance phase contrast objective micro inject as many cells as possible in a rapid fashion. The injected cells can then be examined using the phase contrast to select viable cells. Now wash the injected cells twice with PBS and fix them with 3.7%power of formaldehyde for 30 minutes after fixation.
Wash the cells three times with PBS and then incubate them with PBS supplemented with 0.2%NP 40 for five minutes. Next, block the cells with 4%goat serum in PBS at room temperature for one hour. After blocking, incubate the cells with the desired primary antibody at the appropriate dilution with 1%goat serum in PBS for another hour.
Now wash the cells three times for 10 minutes each time with sodium chloride and triss, and then incubate the cells with the desired fluorescent labeled secondary antibody at a one to 1000 dilution in 1%goat serum in PBS for one hour at 37 degrees Celsius. Finally, wash the cells three times with TBS buffer, using a laser scanning confocal microscope with a high numerical aperture objective set the acquisition parameters, for example, for calcium green, use a 4 88 nanometer excitation and long pass five 15 emission filters for fast calcium measurements. Adjust the pixel time to two microseconds per pixel and set the image zoom to achieve a pixel size of 0.05 to 0.3 microns.
Then select a line from a cell of choice in a particular region in the time controller window. Set the acquisition time for at least 1, 500 scans and record the background reading prior to the stimulation. Keeping the cells in one milliliter of LI of its 15 medium.
Dilute the carbahol to a final concentration of 10 micromolar in lebovits 15. And then gently add one milliliter of this solution into the dish to stimulate the cells and immediately start recording the data. Next, extract the intensities for each pixel from the recorded image.
Save the intensity data as a table using flu view 1000 software. Finally import the intensity data into a data analysis program and bin the data into around nine to 40 bins for comparison to control experiments and electronic noise. In this first figure, the distribution of cavi in canine adult ventricular cardiomyocytes that have been fixed and stained with an antibody to CAVILON three, the major structural caviola protein in these cells and represented here with red staining is shown.
The distribution of cavilon three from the previous figure was compared to GL for Q expression, which appears here in green as can be seen in this figure. The two proteins co localize further in the right hand graphic. The area in the blue box from the left graphic and the two previous graphics has been magnified to determine how the coupling between cavi link three and GL four QX calcium signals.
Calcium imaging studies were performed on a confocal laser scanning microscope system coupled to an inverted microscope with a 40 times objective. For these experiments, cells were loaded with calcium green here, a line scan of calcium green loaded cardiomyocyte where the pixel dwell time was two milliseconds and the line time was 142 milliseconds is shown. The image is composed of 1, 200 lines.
Each line of the previously shown scan corresponds to the intensity fluctuation of calcium green consisting of 71 points taken over a 142 millisecond range and can be used as a rapid readout for calcium responses. For example, in this figure, the calcium activity of a single line of an individual cardiomyocyte as measured by calcium green fluorescence intensity as a function of time in seconds is shown in the top part of this figure. The representative calcium activity of a single line of an individual unstimulated cardiomyocyte is shown, whereas in the bottom part of the figure, the change in fluorescence activity after addition of five millimolar carpool, which enhances the level of intracellular calcium through G alpha Q-P-L-C-B activity can be observed.
Note that even though the intensity values are similar before and after stimulation, it is clear that the Carle stimulated plot has much broader peaks corresponding to more sustained calcium levels than the narrow fluctuation peaks seen before stimulation. Another example of these fast calcium fluctuations can be observed in this next series of figures. The first graph shows raw line data for stimulated ventricular cardiomyocytes in red, and an average of three line traces is shown in black.
To determine the extent that these calcium fluctuations were mediated by G Alpha Q cavi in three interactions, a peptide that destabilizes cavi in 3G Alpha Q interactions was micro injected. The blue line in this figure shows how the injection of the CAV three peptide eliminated the longer calcium current. The columns of raw data from the previous figure were also imported into Sigma plot, and a histogram analysis was carried out here.
The histogram for the control cells was spread over a large number of bins corresponding to a broad time distribution as depicted in this figure by the red line. In contrast, as seen here depicted by the blue line, the bins for the response of cells where the cavi in 3G Alpha Q interaction was disrupted by injection of the peptide are confined to a narrow bin distribution, suggesting that the presence of the peptide eliminates longer duration fluxes. In these last two pairs of graft, the fast intensity fluctuations of calcium green in adult canine ventricular cardiomyocytes on the left and fresh unstimulated rat neonatal cardiomyocytes that lack cavi on the right are compared here.
The corresponding histograms at the intensities over a three minute period were bend at 35 to view slower calcium events. Note that the neonatal cardiomyocytes, again on the right display fast fluctuations on a flat baseline showing the lack of slower calcium activity while a more structured baseline is seen for the adult cells shown on the left. These differences are most easily observed in the histograms.
Following this procedure, slower single cell calcium measurements and electrophysiological methods can be performed to answer additional questions like, how do this changes ultimately affect calcium channels in the plasma membrane after its development? This technique may pave way for the researchers in the field of pharmacology to study effects of different agents on the specific calcium signaling pathways in living cells. After watching this video, you should have a good understanding on how to carry out microinjection and rapid calcium measurements in cells.
