The overall goal of the following experiment is to study intercellular communication via gap junction channels and HEMI channels by quantification of the spread of the intercellular calcium wave evoked by mechanical stimulation in monolayers of primary corneal endothelial cells. This is achieved by first isolating the primary corneal endothelial cells from fresh bovine eyes. As a second step two to three days following seeding in chambered slides, the confluent monolayer of cells is loaded with the fluorescent calcium sensitive dye flu O 4:00 AM.Next, we apply a controlled and localized mechanical stimulus to one single cell of the monolayer in order to evoke an intercellular calcium wave, which is visualized on the confocal microscope.
The results show the spread of the intercellular calcium wave propagation. Ultimately inhibition of gap junction channels and HEMI channels is used to unravel the properties and regulation of different Conex and and panex and isoforms forming gap junction channels and HEMI channels. The main advantage of mechanical stimulation induced intercellular calcium wave propagation is that it provides simple and reliable method to examine the contribution and properties of connecting and axing channels in a non-invasive manner.
Furthermore, the method relies on the physiological signaling molecules and cascades present in the cellular systems studied. Finally, it is easily adaptable to a variety of cell systems, including primary cells and cell models that ectopically express certain connection or PanIN isoforms. This methods can provide insight into the function and dynamic regulation of connection and PanIN based cap junction channels and HEMI channels by the use of SI RNA mediated knockdown of endogenous connection or panics in isoforms and by the use of selective inhibitors of cap junction channels and HEMI channels.
This system is definitely not limited to both corneal endothelial cells. It's adaptable to virtually any cell type and also more complex tissues. This procedure uses a freshly in nucleated eye from a cow no older than 18 months old and not longer than five minutes postmortem.
That was transported to the laboratory in Earl's balance salt solution and 1%iodine solution at four degrees Celsius. First, transfer the eye to a 100 millimeter by 20 millimeter Petri dish. Next, rinse the eye with EBSS containing 1%iodine.
Then sterilize it with a solution containing 70%ethanol from this point onward. Work in a sterile hood. Carefully dissect the cornea from the eye and place it in a 35 millimeter by 10 millimeter Petri dish containing EBSS with the epithelial cell layer facing upward.
Remove any remaining iris tissue still attached to the cornea carefully. If needed, transfer the cornea to another dish of EBSS with the endothelial cell layer upward and then rinse twice with EBSS. Next, place the cornea with the endothelial layer facing upward into an hourglass and cover with growth medium.
To rinse, remove the medium with a suction pipette. Apply 300 microliters of 0.5 gram per liter tripsin solution to the endothelial layer of the cornea. Place the hourglass containing the cornea in a covered Petri dish and transfer to the incubator for 30 minutes at 37 degrees Celsius and 5%carbon dioxide.
After retrieving the hourglass, gently scraped the endothelial cells away from the cornea with a fire polished hook shaped glass pasture pipette. Aspirate the solution containing the endothelial cells and add it to a 25 centimeter square culture flask containing four milliliters of culture medium. Next, apply 300 microliters of growth medium to the cornea.
Repeat the scraping and add the solution containing the endothelial cells to the culture flask. Repeat this step once more, then place the culture flasks containing the corneal endothelial cells in the growth medium in the incubator at 37 degrees Celsius and 5%carbon dioxide. Two days later, add six milliliters of culture medium and refresh the growth medium every second day.
The cells should be confluent within seven to 10 days of isolation to passage. First, aspirate the culture medium and then rinse twice with EBSS. Then add two milliliters trips in solution to the cells and place the flask in the incubator for three to four minutes.
Then add 12 milliliters of growth medium and pipette the medium three times in and out to disperse the cells. Then count the cells using a hemo, cytometer or automated cell counter. Add 165, 000 cells to two well culture slides.
Next, prepare 75 centimeter square culture flasks for a new passage at a density of 6, 250 cells per centimeter square and add fresh culture medium up to a total volume of 25 milliliters and place the flasks and chamber slides into the incubator. The medium is refreshed every two days and co fluency of the cell layer should be reached within three to four days. First, load the cells in the chamber slide with 10 micromolar flu oh 4:00 AM in phosphate buffered saline for 30 minutes at 37 degrees Celsius with gentle shaking while the cells are incubating in flu oh 4:00 AM couple a glass micro pipette with a tip diameter of less than one micrometer to a piso electro crystals nano positioner, and connect to an amplifier mounted on a micro manipulator.
Set the voltage of the nano positioner to between 0.2 and 1.5 volts. Remove the flu of 4:00 AM solution. Then wash the cells five times with phosphate buffered saline, and then incubate the cells with PBS for at least five minutes at room temperature before measurement.
Excited 488 nanometers with argonne laser and use beam splitter HFT 48. Collect the fluorescent emission at 530 nanometers using a long pass emission filter, LP 5 0 5 with the pinhole set at minimum here an oil immersion 40 x objective is used. Place the chamber slide on the stage of a confocal microscope and find an area of confluence cells.
Next, move the micro manipulator to position the glass micro pipet so that it touches the cell membrane at a 45 degree angle. Operate the micro manipulator to briefly touch less than 1%of the single cell membrane with the glass micro pipette and provoke a one second mechanical stimulation to the cell. Then measure spatial changes in calcium ion concentration following mechanical stimulation with a confocal microscope collect and store images.
Finally, draw a polygonal region of interest to define the total surface area of responsive cells using the software of the confocal microscope. Mechanical stimulation induced calcium transients are shown at different time points in control conditions in bovine corneal endothelial cells by representative pseudo colored fluorescence images, the fluorescence intensities before stimulation are shown in the first image. The white arrow in the second image identifies the mechanically stimulated cell.
The calcium wave propagates to six neighboring cell layers with a total area of cells reached by the wave giving an active area of 62, 870 micrometers squared. The line graph shows the time course of the normalized fluorescence value or NF in the mechanically stimulated cell labeled ms, and the average value of NF in the neighboring cell layers one to five labeled on the graph as MB one to MB five. These images show significant changes in the spread of intercellular calcium waves after treatment with exogenous nucleotides in the graph on the left and after treatment with ecto nucleotide aase inhibitors in the graph on the right in more detail, the graph on the left shows a significant decrease in active area after treatment of bovine corneal endothelial cells with the exogenous Aras Aase six and Aase seven, which hydrolyzed A TP and A DP hereby inhibiting the pericrine intercellular communication pathway.
The asterisk signifies a P value of less than 0.001 in the presence versus absence of ace. The graph on the right shows a significant increase in active area after treatment of BCEC with a selective ecto nucleotide inhibitor, A RL 6 7 1 56 hereby enhancing the pericrine intercellular communication pathway. The asterisk signifies a P value of less than 0.001 in the presence versus absence of a RL Once mastered.
This technique can be done in five to 10 minutes for one experiment in one condition when it's carried out properly. Performing a whole series of experiments from which you can compare different conditions takes one day to draw statistical conclusions. Independent experiments performed on different days and from different batches of isolated cells must be used.
Measuring and quantifying intracellular calcium wave propagation allows the identification and characterization of the cell biological regulation of different connection and ine isoforms as gab junction channels and HEMI channels. I also would like to highlight that this method does not stand by itself, but should be complimented by other molecular and functional approaches ranging from diet transfer to a TP release and electrophysiology.
