This is a simple and effective method for isolating and culturing primary brain pericytes for study of intercellular calcium signaling. The obtained cell culture is a nearly homogenous population of pericytes, and it's straightforward to load the pericytes for calcium imaging. The methods described here should provide other researchers in the field with strong tools for studying pericyte biology, and intracellular signaling in pericytes in vitro.
Begin by thawing one vial of capillaries in a 37 degree Celsius water bath. Once the capillaries have thawed, transfer them to a centrifuge tube with 30 milliliters of DMEM comp, and centrifuge them for five minutes at 500 times G.Then, remove the medium from the tube, and re-suspend the palette in 10 milliliters of fresh DMEM comp. Transfer the suspension to a coated T-75 flask, and allow the capillaries to adhere to the bottom for four to six hours in a 37 degrees Celsius incubator supplied with 10%carbon dioxide.
After the incubation, inspect the flask under a light microscope. Fractions of capillaries should now be attached to the bottom of the flask. On day four after seeding the capillaries, inspect them under the microscope to ensure that they are approximately 60 to 70%confluent.
Aspirate the medium, and gently wash the cells with PBS. Add two milliliters of thawed trypsin-EDTA, and leave the flask in the incubator for one to three minutes, taking the flask out frequently, and observing cell detachment with the microscope. When the endothelial cells start to round up, gently tap the flask to detach them.
When the cells have detached, stop trypsinization by adding 10 milliliters of DMEM comp to the flask. Flush the flask a few times with the medium, and aspirate the endothelial cell suspension, which can now be used for other purposes. Add 10 milliliters of DMEM comp to the flask, and check it under the light microscope to assure that the pericytes are still present and attached to the bottom.
Then, put the flask back into the incubator to allow the pericyte-enriched culture to grow. To seed pericytes into coated 96-well plates, detach them as described in the text manuscript. Aspirate the medium, taking care not to disturb the cell pellet, And re-suspend the pellet in one milliliter of fresh DMEM comp.
Count the cells using a counting chamber, then calculate the volume of suspension that should be added to each well to seed 10, 000 cells per well. Add the cell suspension, then add enough DMEM comp to the cells to achieve a total volume of 200 microliters per well. When ready to load the pericytes with Fura-2 AM calcium indicator dye, take the 96-well plate with the cells out of the incubator, and aspirate the medium from the wells.
Wash the cells twice with assay buffer, and add 100 microliters of loading solution to each well. Wrap the plate with tinfoil to avoid photo bleaching, and incubate it for 45 minutes with 30 RPM shaking at room temperature. After the incubation, aspirate the loading buffer, and wash the cells with the assay buffer twice.
Add 100 microliters of fresh to assay buffer, and leave the cells to incubate for 30 minutes. After the incubation, set the temperature of the plate reader to 37 degrees Celsius, and transfer the 96-well plate with cells to the sample plate position, then place the reagent plate with agonist at the reagent plate position. Start by measuring loading of the cells to ensure equal loading of Fura-2 AM in all wells, then perform the measurements with excitation fluorescence wavelength at 340 to 380 nanometers, and the emission wavelength at 510 nanometers.
Add 50 microliters of agonist, at a speed of 150 microliters per second, from the reagent plate to each well with cells. Mount a cover slip into the cell chamber, and tighten it to avoid leaks. Add DMEM comp and the calculated volume of cell suspension into each chamber, for a final volume of 500 microliters.
Incubate the cell chambers at 37 degrees Celsius and 10%carbon dioxide for six days, or until confluent. Once the cells are confluent, take the cell chambers out of the incubator, and aspirate the medium. Wash the cells twice with assay buffer, and add 500 microliters of loading buffer to each chamber, then incubate them at room temperature for 45 minutes.
After the incubation, aspirate the loading buffer, and wash the cells twice with assay buffer, then add 500 microliters of fresh assay buffer to each chamber and incubate them for another 30 minutes at room temperature, to allow cleavage of the AM ester. Replace the buffer with 500 microliters of fresh assay buffer, and proceed with live imaging at a confocal microscope. Mount the cell chamber on the stage of the microscope as gently as possible to avoid disturbance of the cells.
Set the excitation wavelength at 488 nanometers, emission at 515 nanometers, two minute sequential image acquisition with five second intervals, and an x, y image size of 512 by 512 pixels. Add 300 microliters of 100 millimolar ATP to the cell chamber, and initiate image acquisition. This protocol was used to purify pericytes from bovine brain capillaries.
Cellular outgrowth from the seeded capillaries was imaged over the course of nine days. The capillaries were fully attached to the flask at day one, and by day two, endothelial sprouting was visible. After four days, the cellular outgrowth was distinctive, and the endothelial cells were removed via trypsinization.
Remnants of the capillaries were present after the trypsinization, but disappeared from the flask in the following days. After removal of the endothelial layer, the pericytes were allowed to grow until confluency. On day nine, the pericytes reached approximately 80%confluency, and formed islands.
Addition of ATP to the Fura-2 loaded pericytes resulted in an increase in cytosolic calcium levels. The response occurred immediately after addition of ATP to the pericytes, and declined slowly over the measured time period. The calcium response was also measured with real-time confocal imaging.
Baseline fluorescence was measured, then ATP was added at 64 seconds, and a strong intracellular calcium response was evident. Soon after, the cytosolic calcium compartmentalized in the cells, and a reduction in cell area was visible. By 300 seconds, the cell area heavily reduced, and the fluorescence declined almost to baseline levels.
This method has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP, and are able to contract in vitro.
