The authors wish to acknowledge funding from the Lundbeck Foundation Research initiative on Brain Barriers and Drug Delivery (RIBBDD) and Simon Hougners Family Foundation.

1. Preparation of buffers and solutions for cell culturing
<ol>
	<li>Prepare collagen stock solution by dissolving 5 mg of collagen IV from human placenta in 50 mL of PBS overnight at 4 &#176;C. Aliquot the stock solution into 5 mL portions and store at -20 &#176;C.</li>
	<li>Prepare fibronectin stock solution by dissolving 5 mg of fibronectin in 5 mL of sterile water overnight. Store the fibronectin stocks in aliquots of 500 &#181;L at -20 &#176;C. When thawing, add PBS to a final volume of 50 mL to prepare the work solution and store it at 4 &#176;C.</li>
	<li>Prepare Dulbecco&#39;s Modified Eagle Medium (DMEM) complete medium by adding 50 mL of fetal bovine serum (FBS), 5 mL of MEM nonessential amino acids and 5 mL of penicillin/streptomycin (0.1 g/L streptomycin sulfate and 100,000 U/L penicillin G sodium) to 500 mL of DMEM.</li>
	<li>Prepare 5 mg/mL heparin stock solution by dissolving heparin sodium salt in PBS and pass it through a 0.2 &#181;m filter for sterilization. Store the stock solution at 4 &#176;C.</li>
	<li>Prepare growth medium (GM) immediately before use; mix 10 mL of DMEM-comp and 250 &#181;L of heparin stock solution per T75-flask.</li>
</ol>
2. Isolation of capillaries from fresh bovine brain
NOTE: Bovine brain capillaries are isolated and cultured as previously described (Helms et al.10).
<ol>
	<li>Collect brains from calves, no older than 12 months of age, from a slaughterhouse and bring directly to the lab on ice.</li>
	<li>Remove the meninges and collect all gray matter from the brain using a scalpel. Identify the meninges as the film covering the brain and the gray matter by its gray color.</li>
	<li>Use a 40 mL Dounce tissue grinder to homogenize the gray matter in Dulbecco&#39;s Modified Eagle Medium (DMEM). Fill the slim part of the tissue grinder 1/5 with gray matter suspension and add DMEM until the slim part is filled.</li>
	<li>Separate the capillaries from free cells and smaller tissue pieces by filtration of the homogenate through a 160 &#181;m nylon net filter. Flush the filters with DMEM-comp. Retrieve the capillaries and pool the suspensions into 50 mL centrifugation tubes.</li>
	<li>Resuspend the capillaries in DMEM-comp and add an enzyme mix of DNase I (170 U/mL), collagenase type III (200 U/mL) and trypsin (90 U/mL). Leave the suspension for 1 h in a 37 &#176;C water bath for digestion of the capillaries.</li>
	<li>Run the suspension through a 200 &#181;m mesh filter and resuspend in FBS with 10% dimethyl sulfoxide (DMSO). Freeze the capillaries overnight at -80 &#176;C and move them to liquid nitrogen the day after for long term storage. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Seeding and culturing of bovine capillaries
<ol>
	<li>Day 0: Mix 0.7 mL of collagen IV stock with 6.3 mL of PBS. Add the solution to a T75-flask and leave the flask for 2 h at room temperature (RT) or leave it overnight at 4 &#176;C.</li>
	<li>Remove the collagen solution from the flask and wash three times with PBS.</li>
	<li>Add 7 mL of fibronectin work solution and leave the flask for 30 min at RT. Then, remove the fibronectin solution and seed the capillaries immediately after.</li>
	<li>During the 30 min waiting time, thaw one vial of capillaries in a 37 &#176;C water bath.</li>
	<li>When the capillaries are thawed, transfer immediately to a centrifugation tube with 30 mL of DMEM-comp and centrifuge for 5 min at 500 x g and RT. Remove DMEM-comp from the tube and re-suspend the capillary-pellet in 10 mL of fresh DMEM-comp.</li>
	<li>Transfer the 10 mL suspension to the coated T75-flask and leave the capillaries to adhere to the bottom of the flask for 4-6 h in a 37 &#176;C incubator at 10% CO2. 
	NOTE: The cell growth rate is higher at 10% CO2 rather than the conventional 5% CO2.</li>
	<li>After 4-6 h of incubation inspect the flask under a light microscope. Fractions of capillaries should now be attached to the bottom of the flask (Figure 1, day 0).</li>
	<li>Prepare GM and aspirate the DMEN-comp medium very careful from the capillaries and replace it with 10 mL of freshly made GM.</li>
	<li>Day 2: Remove GM from the capillaries and replace with 10 mL of freshly made GM. Cellular outgrowth from the capillaries should be visible under a light microscope at this point (Figure 1, day 2-3).</li>
</ol>
4. Isolation of primary pericytes from bovine brain capillaries
<ol>
	<li>Day 4: Inspect the capillaries under a light microscope. 
	NOTE: The flask should now be approximately 60-70% confluent to provide an appropriate amount of pericytes (Figure 1, day 4). If this is not the case; replace the GM with 10 mL of fresh medium and leave the flask in the incubator for another day.</li>
	<li>Aspirate the medium and wash the cells gently in PBS.</li>
	<li>Add 2 mL of thawed Trypsin-EDTA for endothelial cells and leave the flask in the incubator for 1-3 min. Take out the flask frequently and observe with the microscope during this time period. 
	NOTE: The endothelial cells should round up and detach from the flask; pericytes should be visible as cells with a &#34;ghost&#34;-morphology and still be attached to the surface of the flask. This is a tricky and important step. It is essential to remove most endothelial cells to avoid contamination of the pericyte monoculture, but prolonged trypsinization can also detach the pericytes. The trypsinization time can vary slightly from time to time, and it is therefore of utmost importance to observe the flask frequently with the microscope during the treatment.</li>
	<li>Gently tap the flask, when the endothelial cells have started to round up, to detach the loosened endothelial cells.</li>
	<li>To stop the trypsinization, add 10 mL of DMEM-comp to the flask. Flush the flask carefully a few times with the medium to remove the endothelial cells. Aspirate the endothelial cell suspension from the flask. The endothelial cells can now be used for other purposes.</li>
	<li>Add 10 mL of DMEM-comp to the flask. Look under the light microscope to assure the pericytes are still present and attached to the bottom. Put the flask back into the incubator to allow the pericyte-enriched culture to grow. 
	NOTE: It is important to observe the culture during the following days. If there is still a fair amount of endothelial cells growing another trypsin-treatment can be performed.</li>
	<li>Allow the pericyte monoculture to grow with change of DMEM-comp. medium every second day. Check the growth of the cells under the light microscope (Figure 1, day 5-8).</li>
</ol>
5. Generation and storage of a monoculture of primary bovine pericytes
<ol>
	<li>Day 8-9: Inspect the capillaries under a light microscope 
	NOTE: The pericytes should now have reached 70-80% confluency and grow in islands in the flask (Figure 1, day 9). If the confluency of the pericytes is less than 70%, allow the cells to grow for another day. The pericytes will not form a complete monolayer as the endothelial cells would.</li>
	<li>Aspirate DMEM-comp and wash the pericytes with 7 mL of PBS.</li>
	<li>Add 2 mL of trypsin-EDTA to the flask and leave it in the incubator for 2-3 min. Place the flask frequently under the light microscope to observe when the pericytes round up and detach from the flask. When the pericytes have started to round up, the flask can be gently tapped to detach the cells.</li>
	<li>Gently tap the flask, when the pericytes have started to round up, to detach the cells.</li>
	<li>Add 10 mL of DMEM-comp to the flask to stop the trypsinization process. Flush the flask a few times with the medium to help detach the last pericytes.</li>
	<li>Transfer the 12 mL cell suspension to a 50 mL centrifugation tube and fill up to 30 mL with DMEM-comp.</li>
	<li>Centrifuge the cell suspension for 5 min at 500 x g and RT. Aspirate the DMEM-comp. carefully without touching the cell pellet. Resuspend the cell pellet in 3 mL of FBS with 10% DMSO.</li>
	<li>Transfer the cell suspension into cryovials; add 1 mL to each, so there will be a total of 3 vials per T75-flask of pericytes. Freeze the pericytes at -80 &#176;C overnight and move them to liquid nitrogen the day after for long term storage. 
	NOTE: Cells may be counted before freezing for a later estimate of survival percentage. The protocol can be paused here.</li>
</ol>
6. Setting up a pericyte monoculture for experiments
<ol>
	<li>Coat a T75-flask with collagen IV and fibronectin using the same procedure as mentioned in section 3.1-3.4.</li>
	<li>While the flask is being coated with fibronectin, thaw one vial of pericytes in a 37 &#176;C water bath.</li>
	<li>Transfer the now thawed pericytes from the cryovial to a centrifugation tube with 30 mL of DMEM-comp. Centrifuge the cell suspension for 5 min at 500 x g, RT.</li>
	<li>Carefully aspirate the medium, leaving the cell pellet at the bottom of the tube. Re-suspend the pellet in 10 mL DMEM-comp.</li>
	<li>Collect and transfer the cell suspension to the coated flask. Leave the flask with pericytes to grow in a 37 &#176;C incubator at 10% CO2.</li>
	<li>Every second day, refresh the medium with 10 mL of fresh DMEM-comp. 
	NOTE: After 5 days of growth, the pericytes should have reached approximately 80% confluency. If the confluency is less, leave the cells to grow for another day or two. The cells should now be ready for seeding for further experiments.</li>
</ol>
7. Seeding of pericytes in a coated 96-well plate
<ol>
	<li>Dilute collagen IV as described in step 3.1. Add 100 &#181;L to each well in a 96-well plate and incubate for 2 h at RT or overnight at 4 &#176;C.</li>
	<li>Aspirate the solution and wash the wells three times with PBS.</li>
	<li>Add 100 &#181;L of diluted fibronectin to each well and incubate at RT for 30 min. Remove the fibronectin solution and use the plate immediately. 
	NOTE: Depending on how well the pericyte batch is growing, there should be enough cells for seeding two plates.</li>
	<li>Take out the pericytes from the incubator and aspirate the medium. Wash the cells with PBS.</li>
	<li>Add 2 mL of trypsin-EDTA to the pericytes and follow same procedure as in step 5.3-5.6.</li>
	<li>Aspirate the medium, without harming the cell pellet and re-suspend the pellet in 1 mL of fresh DMEM-comp.</li>
	<li>Take out 12 &#181;L of cell suspension and add to a counting chamber. Under the light microscope, count at least 3 of 3x3 grids and use the average cell count per grid.</li>
	<li>Use the equation below to calculate the volume of cell suspension that should be added to each well to seed 10.000 cells per well, in the 96-well plate. 
	<img src="/files/ftp_upload/61253/61253eq1v2.jpg" /></li>
	<li>Add DMEM-comp and the calculated volume of cell suspension in each well to reach a final volume of 200 &#181;L.</li>
	<li>Place the 96-well plate in a 37 &#176;C incubator at 10% CO2. Leave the cells to grow for 4 days with a change of medium after 2 days.</li>
</ol>
8. Preparation of buffers and solutions for Ca2+-imaging
<ol>
	<li>Autoclave coverslip cell chambers and coverslips.</li>
	<li>Assay buffer: Add 1.19 g of HEPES to 500 mL of HBSS buffer for a final concentration of 10 mM HEPES. Adjust the pH to 7.4.</li>
	<li>Prepare 20% (w/v) Pluronic F127 + 1% (v/v) polyethoxylated castor oil stock solution by dissolving 0.5 g of Pluronic F127 solution in 2.5 mL of anhydrous DMSO in a glass vial. Heat to 40 &#176;C for approximately 30 min or until dissolved and vortex. Add 25 &#181;L of polyethoxylated castor oil and store at RT. Do not freeze.</li>
	<li>Prepare 2 mM Fura-2 AM stock by dissolving 1 mg in 500 &#181;L of anhydrous DMSO. Store in aliquots of 20 &#181;L at -20 &#176;C protected from light.</li>
	<li>Prepare 5 &#181;M Fura-2 AM loading solution by mixing 20 &#181;L of 20% Pluronic F-127 + 1% polyethoxylated castor oil stock solution with the 20 &#181;L of 2 mM Fura-2 AM aliquot. Add 500 &#181;L of assay buffer and vortex. Add assay buffer to a final volume of 8 mL. The solution should be prepared immediately before use and protected from light.</li>
	<li>Prepare 4 mM Cal-520 AM by dissolving 1 mg in 226.7 &#181;L of anhydrous DMSO. Store in aliquots of 20 &#181;L at -20 &#176;C protected from light.</li>
	<li>Prepare 20 &#181;M Cal-520 AM loading solution by mixing 20 &#181;L of 20% Pluronic F-127 + 1% polyethoxylated castor oil stock solution with the 20 &#181;L 4 mM Cal-520 aliquot. Add 500 &#181;L of assay buffer and vortex. Add assay buffer to a final volume of 4 mL. The solution should be prepared immediately before use and protected from light.</li>
</ol>
9. Loading of pericytes with Fura-2 AM calcium indicator dye in a plate-reader setup
NOTE: All solutions should be at RT before the experiment starts.
<ol>
	<li>Take out the 96-well plate with cells from the incubator and aspirate the medium from the wells. Wash the cells twice with assay buffer.</li>
	<li>Add 100 &#181;L of loading solution to each well and wrap the plate with tinfoil, to avoid photo bleaching. Incubate for 45 min with 30 rpm shaking at RT. 
	NOTE: Do not load Fura-2 AM at 37 &#176;C, as it may load internal compartments. Remember to leave wells with cells in assay buffer instead of loading buffer; these are&#160;the &#34;blanks&#34; used for measuring background fluorescence.</li>
	<li>Aspirate the loading buffer and wash the cells with assay buffer twice. Add 100 &#181;L of fresh assay buffer and leave the cells to incubate for 30 min at RT; this allows for continuous cleavage of the AM-ester and thereby trapping Fura-2 AM inside the cells.</li>
	<li>Prior to the Ca2+-imaging, wash and replace the buffer with 100 &#181;L of fresh assay buffer.</li>
</ol>
10. Well-plate fluorescence reading of pericytes in a plate-reader setup
<ol>
	<li>Set the temperature of the plate reader to 37 &#176;C and transfer the 96-well plate with cells to the sample plate position. Place the reagent plate with agonist at the reagent plate position.</li>
	<li>Start by measuring loading of the cells to ensure equal loading of Fura-2 AM in all wells.</li>
	<li>Perform the measurements with excitation fluorescence wavelength at 340 nm/380 nm and the emission wavelength at 510 nm. Add 50 &#181;L of agonist at speed 150 &#181;L/s from the reagent plate to each well with cells in the sample plate position.</li>
	<li>Save the data and export as xlsx files for further analysis. Figure 2 shows the cytosolic Ca2+-response measured as the ratio between the two excitation wavelengths over time, where background fluorescence is subtracted. 
	NOTE: The plate-reader need to be a dual microplate reader with room for a &#34;cell tray&#34; and a &#34;sample tray&#34; and an integrated pipettor system.</li>
</ol>
11. Seeding of pericytes in a coated cell chamber for live imaging
NOTE: Coverslips may also be placed in the bottom of culture wells, coated and seeded with pericytes as described above, and then mounted in the chamber prior to experiments.&#160;&#160;
<ol>
	<li>Mount a coverslip into the cell chamber and make it tight to avoid leakiness.</li>
	<li>Dilute collagen IV as described in step 3.1. Add 500 &#181;L to each cell chamber and incubate for 2 h at RT or overnight at 4 &#176;C.</li>
	<li>Aspirate the collagen solution and wash the chambers three times with 500 &#181;L of PBS.</li>
	<li>Add 500 &#181;L of diluted fibronectin to each well and incubate at RT for 30 min. Remove the fibronectin solution and use the cell chamber straight afterwards.</li>
	<li>In the meantime, take out the flask with confluent pericytes and wash with 7 mL of PBS.</li>
	<li>Add 2 mL of trypsin-EDTA to the pericytes and follow same procedure as in step 5.3-5.6.</li>
	<li>Proceed by following the same steps as in step 8.6-8.7.</li>
	<li>Use the equation below to calculate the volume of cell suspension, which should be added to each chamber to seed 90.000 cells per chamber. 
	<img src="/files/ftp_upload/61253/61253eq2v2.jpg" /></li>
	<li>Add DMEM-comp and the calculated volume of cell suspension in each chamber to reach a final volume of 500 &#181;L.</li>
	<li>Place the cell chambers in the incubator at 37 &#176;C, 10% CO2. Leave the cells to grow for 6 days (or until confluent). 
	NOTE: The pericytes grow slower on glass-slides compared to plastic; more days of growth are necessary.</li>
</ol>
12. Loading of pericytes with Cal-520 AM calcium indicator dye for live imaging
NOTE: All solutions should be at RT before the experiment starts.
<ol>
	<li>Prepare the 20 &#181;M Cal-520 AM loading buffer: Mix 20 &#181;L of 20% Pluronic F-127 + 1% polyethoxylated castor oil stock solution with the 20 &#181;L 4 mM Cal-520 aliquot. Add 500 of &#181;L assay buffer and vortex. Add assay buffer to a final volume of 4 mL. The solution should be prepared immediately before use and protected from light. 
	NOTE: Protect solutions containing Cal-520 AM from light exposure.</li>
	<li>Take out the cell chambers from the incubator and aspirate the medium. Wash the cells twice with assay buffer.</li>
	<li>Add 500 &#181;L of loading buffer to each chamber and incubate at RT for 45 min.</li>
	<li>Aspirate the loading buffer and wash the cells twice with assay buffer.</li>
	<li>Add 500 &#181;L of fresh assay buffer to each chamber and incubate for 30 min at RT to allow cleavage of the AM-ester.</li>
	<li>Replace the buffer with 500 &#181;L of fresh assay buffer before performing the live imaging at a confocal microscope.</li>
</ol>
13. Live imaging of intracellular Ca2+-levels
NOTE: A variety of microscope types can be used for the imaging. Upright or inverted conventional fluorescence microscopes, as well as upright or inverted confocal laser scanning microscopes with appropriate excitation source (488 nm) and emission filters (510-520 nm) can be used. Objectives should be suited for fluorescence and be of a high quality and with high numerical aperture (NA).
<ol>
	<li>Mount the cell chamber on the stage of the confocal microscope as gentle as possible, in order to avoid disturbance of the cells.</li>
	<li>Select an excitation wavelength of 488 nm, emission at 515 nm, sequential image acquisition with 5 second intervals, an XY image size of 512 x 512 pixels and measure for 2 min to measure baseline calcium signals.</li>
	<li>Add 3 &#181;L of 100 mM ATP to the cell chamber with a pipette, and continue the sequential image acquisition. Perform the addition slowly and gently to not disturb the preparation and move the cells out of focus.</li>
	<li>Observe the degree of changes and increase the time interval over time as needed for approximately 18 min until no further morphological change is noted (Figure 3).</li>
	<li>Save time-lapse images and export them as TIFF and/or AVI files for further analysis. 
	NOTE: One vial of pericytes should give enough cells for seeding in 1-2 96-well plates and several coverslips, meaning you can prepare cells for both types of calcium-measurements.</li>
</ol>

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The authors declare no competing financial interests.

In this study, we have presented a method to isolate primary pericytes from bovine brains. The described protocol allows culture of this otherwise rather inaccessible cell type. The subsequently obtained cell culture was a nearly homogenous population of pericytes, with little or no contamination with endothelial cells&#160;and glial cells based on cell morphology and protein expression12. Furthermore, we demonstrated a simple and straightforward method to load the pericytes with calcium dyes for ...

Brain capillary pericytes, together with endothelial cells and astrocytes, constitute the NVU1,2,3. The endothelial cells, which form the structural basis of the capillaries, form long cylindrical tubes with a diameter of 5-8 &#181;m. The endothelial cells are sporadically covered with pericytes and surrounded by protrusions from astrocytes; the astrocyte endfeet.
The blood-brain barrier (BBB), situated at the brain capillaries, is the main site for exchange of nutrients, gases and waste products between the brain and the blood. The BBB also protects the brain from endogenous and exogenous neurotoxins and serves as a barrier for the delivery of a large number of drug compounds. The barrier function is a focus area, as well as an obstacle, for drug companies developing central nervous system (CNS) medicines. This has spurred a large interest in investigating the cells of the NVU in culture4. Brain astrocytes and endothelial cells have been cultured and characterized in a number of studies, whereas the studies and protocols for pericyte culture are sparse.
Previously published protocols have described generation of brain capillary pericyte cultures to some degree, using a range of different approaches such as immunopanning5, high- and low-glucose media6, fluorescent-activated cell sorting7, density gradient centrifugation8, etc. Although these methods seem sufficient to obtain cultures of pericytes, some are time consuming, cost expensive and the pericytes obtained might not be ideal due to the number of culture passages that can de-differentiate the pericytes9. Furthermore, the potential of cultured pericytes in in vitro signaling studies has been fairly unexplored until now.
The present work focuses on the generation of pericyte cultures from isolated bovine brain capillaries and the subsequent setup for measurements and imaging studies of changes in intracellular calcium, an important intracellular second messenger. We briefly describe the isolation of capillaries from cortical gray matter (for details see Helms et al.10) and the isolation and culture of pericytes in pure monoculture without contamination with endothelial or glial cells. We then provide a protocol for seeding of pericytes in 96-well plates and loading protocols for the calcium probe Fura-2 AM. Finally, we show how pericytes can be used in real-time confocal imaging in microscope culture chambers and describe the protocols for this.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ATP</td>
			<td>Tocris</td>
			<td>3245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cal-520 AM</td>
			<td>AAT Bioquest</td>
			<td>21130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Standard large volume centrifuge for spinning down cells</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 510</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>FastRead</td>
			<td>102</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip cell chamber</td>
			<td>Airekacells</td>
			<td>SC15022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cremophor EL</td>
			<td>Sigma Aldrich</td>
			<td>C5135</td>
			<td>Formerly known as Kolliphor EL</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>471267</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium</td>
			<td>Sigma Aldrich</td>
			<td>D0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA/GE Healthcare</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Thermo Fisher</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 22x22 mm</td>
			<td>VWR International</td>
			<td>631-0123</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem Panreac</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>Olympus CK2</td>
			<td>Upright light microscope with phase contrast</td>
		</tr>
		<tr>
			<td>MEM nonessential amino acids</td>
			<td>Sigma Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>BMG LabTech</td>
			<td>NOVOstar</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>penicillin G sodium/streptomycin sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4299</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Sigma Aldrich</td>
			<td>CLS3972</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning incorporated</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here 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.

Bovine brain capillaries were isolated from fresh brain tissue and Figure 1 presents the capillary seeding and cellular outgrowth over days and subsequent purification&#160;of pericytes. The capillaries are fully attached to the flask at day 1 and on day 2 endothelial sprouting has become visible (Figure 1, day 2). After 4 days, the cellular outgrowth is highly distinctive (Figure 1, day 4a) and the ...

Watch this Scientific Journal Video about Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies at JoVE.com

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

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4.7K Views.  University of Copenhagen. Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Video: Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...