LT and FG were granted by Agence Nationale de la Recherche (ANR, ANR-15-JPWG-0010) in the framework of the EU Joint Programme &#8211; Neurodegenerative Disease Research (JPND) for project SNOWBALL.

All experiments were performed following the Institute&#39;s guidelines for the animal use and handling. In accordance with the French legislation, the animal facility at the University of Artois has been approved by the local authorities (reference: B62-498-5). In compliance with the European Union Legislation (Directive 2010/63/EU), all the procedures were approved by the local animal care and use committee (Comit&#233; d&#39;Ethique en Exp&#233;rimentation Animale du Nord-Pas-De-Calais, reference: C2EA 75) and the French Ministry of Research (reference: 2015090115412152).
1. Preparation of solutions
<ol>
	<li>Prepare 500 mL of Washing Buffer A (WBA): 10 mM HEPES solution in Hank&#39;s Balanced Salt Solution (HBSS). Store at 4 &#176;C.</li>
	<li>Prepare 500 mL of Washing Buffer B (WBB): 0.1% Bovine Serum Albumin (BSA) with 10 mM HEPES solution in HBSS. Store at 4 &#176;C.</li>
	<li>Prepare 300 mL of 30% dextran solution in WBA by mixing the solution overnight at room temperature. Autoclave the solution at 110 &#176;C for 30 min before use. After autoclaving, let the solution rest at room temperature for 2-3 h. Store the solution at 4 &#176;C.</li>
	<li>Prepare 100 mL of 0.1% BSA solution in cold dextran solution. Shake vigorously for 3-4 min and store at 4 &#176;C.</li>
	<li>Prepare complete Dulbecco&#39;s Modified Eagle Medium (DMEM) culture media by dissolving 20% calf serum, 2 mM glutamine, 50 &#181;g/mL gentamycin, 1% vitamins, 2% amino acids Basal Medium Eagle (BME) in Basal DMEM media and store at 4 &#176;C. Add 1 ng/mL Basic fibroblast growth factor (bFGF) prior to use.</li>
	<li>Prepare complete pericyte media by adding pericyte growth supplements (provided with the pericyte media) and 20% Fetal Calf Serum (FCS) in pericyte culture basal media and store at 4 &#176;C.</li>
</ol>
2. Brain tissue recovery and removal of meninges
<ol>
	<li>For consistency, use mice of similar age and same gender in every batch of extraction. Use a pathogen-free animal shelter and provide ad libitum access to water. To ensure efficiency and minimal use of animals, avoid loss of tissue material.</li>
	<li>Euthanize C57BL/6J, 4-6 weeks old, male mice (Janvier labs, Le Genest-Saint-Isle, France).</li>
	<li>Quickly excise the brain tissue in sterile conditions, avoid any damage to the tissue. Carefully place the tissue in 40 mL of cold phosphate buffered saline (PBS).</li>
	<li>Transfer the brain tissue in cold PBS to a Petri dish (100 mm x 15 mm).</li>
	<li>Place the brain tissue on a sterile dry lint-free wipe and with curved tip forceps, remove the cerebellum, striatum and occipital nerves.
	<ol>
		<li>Remove all the visible meninges with a cotton swab. Place the brain tissue upside down and open the lobes with a cotton swab using outward light strokes. Remove all the visible blood vessels.</li>
		<li>Place the meninges free brain tissue in a Petri dish (100 mm x 15 mm) with 15 mL of cold WBB.</li>
	</ol>
	</li>
</ol>
3. Homogenization
<ol>
	<li>Transfer the tissue to a Dounce tissue grinder mortar tube and add 3-4 mL of WBB with forceps.</li>
	<li>Mince the tissue with a &#39;loose&#39; pestle 55 times. Rinse the &#39;loose&#39; pestle with WBB. Now mince the slurry with a &#34;tight&#34; pestle 25 times.</li>
	<li>Divide the slurry equally into two 50 mL tubes and add 1.5x volume of cold 30% BSA-dextran, vigorously shaking the tubes to mix the slurry.</li>
</ol>
4. Isolation of the vascular fraction
<ol>
	<li>After vigorously shaking the tubes, centrifuge the tubes for 25 min at 3,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant (along with the top myelin layer) to 2 new tubes, and centrifuge the tubes for 25 min at 3,000 x g and 4 &#176;C. Preserve the pellets from the first centrifugation by adding 3 mL of cold WBB (keep the pellet at 4 &#176;C).</li>
	<li>Repeat step 4.2 and carefully preserve the pellets from 2nd centrifugation.</li>
	<li>Discard the dextran and the myelin with tissue debris from the tubes from step 4.3. Preserve the pellets in cold WBB.</li>
	<li>Pool the contents of tube 1 and tube 2 from step 4.1 and make up to final volume of 10 mL with cold WBB.</li>
	<li>Repeat this step for tubes from step 4.2 and step 4.3. 
	NOTE: Finally, there are 3 tubes from 3 centrifugations.</li>
	<li>Dissociate the pellet with 6 up and down strokes using a 10 mL pipette, until no visible clumps of the pellets are remaining.</li>
	<li>With the help of a vacuum filter assembly and the nylon mesh filter, filter the cell suspensions of each tube. 
	NOTE: This filtration step is important in order to remove longer/larger vessels via the mesh filter.</li>
	<li>Recover and resuspend the capillaries by scraping the filter with the help of a flat tip forceps or a scraper in WBB at room temperature. Filter the suspension with a fresh filter to recover more capillaries</li>
	<li>Divide the filtrate equally into two tubes and centrifuge for 7 min at 1,000 x g and RT. 
	NOTE: During this centrifugation step, prepare the enzymatic solution. Determine the volume of WBB required in accordance with the number of animals used (see Table of Materials). Add 1x of DNase 1 and 1x of Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) (see Table of Materials) in WBB and pre-warm to 37 &#176;C.</li>
	<li>Collect the pellets from step 4.10 in one tube with pre-warmed WBB with enzymes. Add pre-warmed 1x collagenase/dispase. Place the tube in the shaking table water bath at 37 &#176;C for precisely 33 min.</li>
	<li>Stop the enzyme reaction by adding 30 mL of cold WBB. Centrifuge the suspension for 7 min at 1,000 x g and RT.</li>
	<li>Discard the supernatant carefully and dissociate the pellet in WBB with 6 up and down strokes using a 10 mL pipette. This step should be less rigorous and comparatively faster.</li>
	<li>Centrifuge the suspension for 7 min at 1,000 x g and RT. 
	NOTE: During this step, discard the coating from the culture dishes and rinse them once with DMEM at room temperature. Coat cell culture dishes for at least 1 h at RT.</li>
	<li>Discard the supernatant from the tube obtained at step 4.14, and dissociate the pellet in new complete DMEM media, plate the cells (Day 0, P0) in 9 wells of 6-well plates (1 well of 9.6 cm2 each).</li>
</ol>
5. Proliferation of cerebral pericytes
<ol>
	<li>Maintain the cell cultures at 37 &#176;C and 5% CO2 in a sterile incubator. Replace the culture media after 24 h (day 1) of plating the cells by carefully removing the debris. After day 1, change the culture media in every 48 h.</li>
	<li>Observe the cell culture for at least 7-8 days. By this time, cellular growths on the top of endothelial unilayer should be observable.</li>
	<li>Passage the cells on day 8-10 (depending on the confluency) in pericyte culture medium to passage 1 (P1) on gelatin coated culture plates. Change the culture media in every 2 days. Observe the cells for 6-7 days. Cells are consecutively split again to passage 2 (P2) on day 17 [and passage 3 (P3) on day 24 only if required], grown in pericyte medium in gelatin coated plates. 
	NOTE: Cells shall be ready for experiments/observation at nearly 80-90% confluency.</li>
</ol>

<ol>
	<li>Azevedo, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+modulate+myelination+in+the+central+nervous+system.">Pericytes modulate myelination in the central nervous system.</a> Journal of Cellular Physiology. 233 (8), 5523-5529 (2018).</li><li>Hall, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+pericytes+regulate+cerebral+blood+flow+in+health+and+disease.">Capillary pericytes regulate cerebral blood flow in health and disease.</a> Nature. 508 (7494), 55-60 (2014).</li><li>Gianni-Barrera, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-BB+regulates+splitting+angiogenesis+in+skeletal+muscle+by+limiting+VEGF-induced+endothelial+proliferation.">PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.</a> Angiogenesis. 21 (4), 883-900 (2018).</li><li>Teichert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte-expressed+Tie2+controls+angiogenesis+and+vessel+maturation.">Pericyte-expressed Tie2 controls angiogenesis and vessel maturation.</a> Nature Communications. 8, 16106 (2017).</li><li>Dave, J. M., Mirabella, T., Weatherbee, S. D., Greif, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+ALK5/TIMP3+Axis+Contributes+to+Endothelial+Morphogenesis+in+the+Developing+Brain.">Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain.</a> Developmental Cell. 44 (6), 665-678 (2018).</li><li>Franco, M., Roswall, P., Cortez, E., Hanahan, D., Pietras, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+promote+endothelial+cell+survival+through+induction+of+autocrine+VEGF-A+signaling+and+Bcl-w+expression.">Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression.</a> Blood. 118 (10), 2906-2917 (2011).</li><li>Saint-Pol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Pericytes+ABCA1+Expression+Mediates+Cholesterol+Efflux+but+not+Cellular+Amyloid-beta+Peptide+Accumulation.">Brain Pericytes ABCA1 Expression Mediates Cholesterol Efflux but not Cellular Amyloid-beta Peptide Accumulation.</a> Journal of Alzheimer's Disease. 30 (3), 489-503 (2012).</li><li>Sagare, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+loss+influences+Alzheimer-like+neurodegeneration+in+mice.">Pericyte loss influences Alzheimer-like neurodegeneration in mice.</a> Nature Communications. 4, 2932 (2013).</li><li>Montagne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+degeneration+causes+white+matter+dysfunction+in+the+mouse+central+nervous+system.">Pericyte degeneration causes white matter dysfunction in the mouse central nervous system.</a> Nature Medicine. 24 (3), 326-337 (2018).</li><li>Claudio, L., Raine, C. S., Brosnan, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+persistent+blood-brain+barrier+abnormalities+in+chronic-progressive+multiple+sclerosis.">Evidence of persistent blood-brain barrier abnormalities in chronic-progressive multiple sclerosis.</a> Acta Neuropathologica. 90 (3), 228-238 (1995).</li><li>Nishioku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+of+brain+pericytes+from+the+basal+lamina+is+involved+in+disruption+of+the+blood-brain+barrier+caused+by+lipopolysaccharide-induced+sepsis+in+mice.">Detachment of brain pericytes from the basal lamina is involved in disruption of the blood-brain barrier caused by lipopolysaccharide-induced sepsis in mice.</a> Cellular and Molecular Neurobiology. 29 (3), 309-316 (2009).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Functions+and+Mechanisms+of+Pericytes+in+Ischemic+Stroke.">Diverse Functions and Mechanisms of Pericytes in Ischemic Stroke.</a> Current Neuropharmacology. 15 (6), 892-905 (2017).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PDGF-BB-SOX7+axis-modulated+IL-33+in+pericytes+and+stromal+cells+promotes+metastasis+through+tumour-associated+macrophages.">The PDGF-BB-SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages.</a> Nature Communications. 7, 11385 (2016).</li><li>Hesp, Z. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferating+NG2-Cell-Dependent+Angiogenesis+and+Scar+Formation+Alter+Axon+Growth+and+Functional+Recovery+After+Spinal+Cord+Injury+in+Mice.">Proliferating NG2-Cell-Dependent Angiogenesis and Scar Formation Alter Axon Growth and Functional Recovery After Spinal Cord Injury in Mice.</a> Journal of Neuroscience. 38 (6), 1366-1382 (2018).</li><li>Guo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor-B+enhances+glioma+angiogenesis+by+stimulating+vascular+endothelial+growth+factor+expression+in+tumor+endothelia+and+by+promoting+pericyte+recruitment.">Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment.</a> American Journal of Pathology. 162 (4), 1083-1093 (2003).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+pericytes+for+therapeutic+approaches+to+neurological+disorders.">Targeting pericytes for therapeutic approaches to neurological disorders.</a> Acta Neuropathologica. 136 (4), 507-523 (2018).</li><li>Tigges, U., Welser-Alves, J. V., Boroujerdi, A., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+culturing+pericytes+from+mouse+brain.">A novel and simple method for culturing pericytes from mouse brain.</a> Microvascular Research. 84 (1), 74-80 (2012).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD146+coordinates+brain+endothelial+cell-pericyte+communication+for+blood-brain+barrier+development.">CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (36), 7622-7631 (2017).</li><li>Thomsen, M. S., Birkelund, S., Burkhart, A., Stensballe, A., Moos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+deposition+of+basement+membrane+proteins+by+primary+brain+capillary+endothelial+cells+in+a+murine+model+of+the+blood-brain+barrier.">Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.</a> Journal of Neurochemistry. 140 (5), 741-754 (2017).</li><li>Yamazaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Cell+Senescence+Contributes+to+Blood-Brain+Barrier+Breakdown.">Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown.</a> Stroke. 47 (4), 1068-1077 (2016).</li><li>Crouch, E. E., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+isolation+of+endothelial+cells+and+pericytes+from+mouse+brain+microregions.">FACS isolation of endothelial cells and pericytes from mouse brain microregions.</a> Nature Protocols. 13 (4), 738-751 (2018).</li><li>Ozerdem, U., Grako, K. A., Dahlin-Huppe, K., Monosov, E., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2+proteoglycan+is+expressed+exclusively+by+mural+cells+during+vascular+morphogenesis.">NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.</a> Developmental Dynamics. 222 (2), 218-227 (2001).</li><li>Stallcup, W. B., You, W. K., Kucharova, K., Cejudo-Martin, P., Yotsumoto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2+Proteoglycan-Dependent+Contributions+of+Pericytes+and+Macrophages+to+Brain+Tumor+Vascularization+and+Progression.">NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression.</a> Microcirculation. 23 (2), 122-133 (2016).</li><li>Armulik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+regulate+the+blood-brain+barrier.">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li></ol>

The authors have nothing to disclose.

Cerebral pericytes are an integral part of the NVU and play an active role in induction and maintenance of the BBB24. Similarly, the role of these cells in the different neurodegenerative disorders and vascular pathologies is intriguing. Hence, an efficient high output primary pericyte cell model will provide an efficient platform for in vitro studies.
There are various protocols that have been proposed for the isolation of primary pericytes (Figure 4). Tigges et al.17 suggested a method including cortical tissue with meninges. This approach is tenderization of tissue from 6 mice (a 37 &#176;C, 70 min digestion with papain/DNase enzymes) followed by a disintegration step via 21 G and 18 G needles. This protocol suggests a one-step separation (centrifugation in 22% BSA/PBS solution) that yields at least 2 collagen I coated wells of a 6-well plate. The cells are maintained in endothelial cell growth medium (ECGM) until passage 3 and later in pericyte growth medium (PGM) for passaging cells to promote pericyte proliferation. In another similar approach, Chen et al.18 proposed tissue dissociation by dicing the tissue with a sterilized razor blade and tissue digestion with collagenase/DNase for 90 min at 37 &#176;C. Following one-step separation (centrifugation in 22% BSA) of the cells, the myelin layer is removed and the pellet is washed twice in ECGM. The microvessels are plated in 3 wells of a collagen I coated 6-well plates. After reaching confluence, the cells are passaged twice and later maintained in PGM. In the end, if cells are passed in ratio of 3, we can obtain 27 wells of 6-well plates only at the use of 10 mice in the beginning of the protocol.
In Thomsen et al., the authors suggest isolation of cerebral pericytes via a two-step enzyme digestion19. Meninges and white matter are removed, and brain samples are cut into small pieces. The tissue pieces undergo the first enzyme reaction in collagenase/DNase I for 75 min at 37 &#176;C, following one step of separation in 20% BSA. The pellet is collected and further digested in collagenase/dispase/DNase I for 50 min at 37 &#176;C. This step is followed by microvessel separation in a 33% Percoll gradient and further washed once. The microvessels are seeded on collagen IV/fibronectin coated 35 mm dishes. The proliferation of pericytes is favored by 10% FCS and gentamicin sulphate in DMEM for 10 days. In another two-step enzyme digestion approach, Yamazaki et al. suggest mincing of the excised tissue in cold DMEM20. In the first enzyme reaction, samples are treated with collagenase/DNase I for 75 min at 37 &#176;C. Following one step centrifugation, the pellet is again washed once and a second enzyme reaction is initiated in collagenase/dispase for 60 min at 37 &#176;C. Following a one-step separation, the pellet is resuspended and centrifuged in 22% BSA solution. Finally, the microvascular pellet is resuspended and plated in a 6-well plate. For 5 mouse brains, 1 well of a 6-well plate can be plated. To obtain the pericytes, endothelial cultures are passaged thrice while maintained in mouse brain endothelial cell (mBEC) medium II. Crouch and Doetsch20 suggest pericyte purification method by FACS. Tissue samples from cortex and ventricular&#8211;subventricular zone of mouse brain are micro-dissected and minced thoroughly with a scalpel. After collagenase/dispase enzyme incubation for 30 min at 37 &#176;C, the digested tissue is separated from myelin and debris centrifuged in a 22% v/v Percoll solution. The cell suspension is then incubated in fluorescently conjugated antibodies for FACS analysis and sorting. The sorted cells are plated in collagen coated wells of 24 well plate. It is suggested that one cortex yields enough cells for one plating in 1 well of 24-well plate.
Even if productive, these methods come with several limitations, from the usage of high number of animals for single batch isolation to a very limited amount of output.
During the development of this proposed protocol, we were successful in obtaining high output: 9 wells of a 6-well plate from as few as 10 mice. To this end, removal of meninges ensures the one step removal of large vessels from the tissue. The Dounce tissue grinder is more appropriate for soft tissues such as the brain. It also ensures sample reduction with the loose pestle, homogenization with the tight pestle, and prevents unnecessary cellular damage. One of the main objectives in primary cell culture protocols is the minimal waste of tissue and extended retrieval of cerebral vasculature. In the presented protocol, this is achieved by repetitive centrifugation of the dextran-BSA infused tissue homogenate. A three-step centrifugation approach helps to recover large quantities of vasculature from the tissue homogenate. This provides a 3x enhanced recovery of microvessels. Following separation, filtration is the next essential step, which favors the exclusion of smooth muscle cell associated large vessels. As mentioned before a combination of different enzymes has been proposed for enzymatic digestion. While DNase and collagenase/dispase are used to reduce clumps of cells and isolate single cells respectively, it is very important to prevent cell death in such an invasive environment and this is prevented by TLCK, which thereby increases the final yield. Initially, the first passage is allowed to grow an endothelial monolayer, which later supports the growth of attached pericytes on the unilayer. Since the survival of primary endothelial cells is reduced upon passaging, it enhances the probability for pericyte retrieval. Moreover, this protocol employs another passaging that ensures avoidance of endothelial cell contamination. It should be noted that with a higher number of cells from P2, the dependency on further passaging of the cells is reduced. In addition, it reduces the possibility of pericyte growth being overtaken by smooth muscle cells, which proliferate on much higher rate.
In order to achieve a higher output, there are several steps that are critical and should be accurately performed with respect to temperature and time. The mixing of tissue homogenate into BSA-dextran should be fast. The pellet dissociation after the centrifugation steps should be quick to prevent cell death. Moreover, 33 min of enzymatic digestion should be done with precision and care. One of the limitations for this protocol is the 7-8 day duration that endothelial unilayer is allowed to grow and further facilitate the growth of pericytes. Evidently, the isolation of microvessels is btter, the growth of unilayer is faster, and hence there are an increased number of pericytes. It is recommended not to use less than 10 mice in each extraction to ensure an adequate number of microvascular fractions to support the pericyte growth further. If the aforementioned points are followed carefully, the desired cell density for cerebral pericyte culture can be easily achieved.
In vitro models provide a feasible platform for the development of derivative models to obtain more information on the pathophysiological relevance and communication among the other cells of the NVU during neurological disorders. Isolated pericytes can be incorporated in a bi- cellular culture (with endothelial or glial cells) and tri-cellular culture (endothelial and glial cells) models. The development of these models has not been discussed here. To conclude, this protocol provides one approach for the isolation of primary cells with higher output and a better platform for in vitro research related to the cerebral pericyte biology.

Cerebral pericytes are an essential component of the neurovascular unit (NVU), which comprises a functional unit with the cerebral endothelial cells of the blood brain barrier (BBB), glial cells, extracellular matrix and neurons. Pericytes are a vital part in regulated functioning of the central nervous system (CNS) as they serve as one of the interfaces for the exchange of molecular and cellular information.
Cerebral pericytes are embedded in the abluminal side of the brain microvessels, and are essential for establishing1 and maintaining2 the BBB physiology. Several recent works have also highlighted the role of cerebral pericytes in angiogenesis3 and vessel maturation4, endothelial morphogenesis5 and survival6, and in controlling the brain cholesterol metabolism7. Importantly, the dysregulation in any of these processes are etiological hallmarks of neurodegenerative diseases.
Indeed, pericytes are a functional necessity for normal BBB functioning and its protection against the progression of several neurological diseases. Degenerating physiology and loss of pericytes are common denominators in the progression of Alzheimer&#39;s disease8, neuronal loss during white matter dysfunction9, multiple sclerosis10, septic encephalopathy11, acute phase ischemic stroke12 and in other neurological disorders. Pericytes are also instrumental in tumor metastasis13. Interestingly, pericytes have also been shown to exhibit a rescuing role after neurological trauma and disorders: in remyelination in brain1, ischemic stroke, spinal cord injury14 and promoting angiogenesis15. The susceptibility of pericytes to reinforce the pathophysiological manifestation of neurological trauma and disorders makes them a potential therapeutic target16.
In vitro research models of pericytes in the BBB are important tools to conduct extensive studies. These models provide a platform for more elaborate studies by representing working models of the BBB and more. For instance, these models can be used to understand the cellular physiology within pericytes and among other cell types of the NVU. Also, in vitro models are firsthand investigation tools for testing the pharmacological influence of new drugs and molecules on pericytes. These models can also be used to understand the pathophysiological role of pericytes in relation to neurological disorders. Nevertheless, the development of in vitro models requires increased output to enable experimental freedom. These models should be easy and quick, and reduce the number of experimental animals used. In addition, the ability to develop such models into a double and triple cell culture models is desirable.
There are many protocols that have been developed. The protocols proposed by Tigges et al.17, Chen et al.18, Thomsen et al.19, Yamazaki et al.20, and Crouch and Doetsch21 are commendable approaches that satisfy most of the necessities. All of these methods yield effective results, but the dependency on a large number of experimental animals remains a common denominator for these protocols. Therefore, it becomes mandatory to develop a high output method that can isolate and purify pericytes with maximum possible efficiency. In this protocol, the purity of the cells obtained after a second passage is verified with several pericytes markers. We checked for Platelet-Derived Growth Factor Receptor-&#946; (PDGFR- &#946;), which is used as a classical marker of pericytes17, and for NG2 (neuron-glial antigen 2), which is a marker of pericyte mediated vascular morphogenesis22 and vascularization23. We also checked for cluster of differentiation 146 (CD 146), which has been reported as one of the molecules expressed in the pericytes17,18.
Here, we present a protocol for the extraction of primary pericytes from mice (wild type or transgenics) that will satisfy all the aforementioned requirements with high output. We employ an antibiotic and immunopanning free selection-based method of proliferation for the primary cerebral pericytes, which will prove itself an efficient model for conducting in vitro studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino acids BME</td>
			<td>Sigma</td>
			<td>B-6766</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Basal DMEM media</td>
			<td>Invitrogen</td>
			<td>316000083</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor</td>
			<td>Sigma</td>
			<td>F-0291</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8412</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Collagenase dispase</td>
			<td>Sigma</td>
			<td>10269638001</td>
			<td>Prepare a 10x stock solution in sterile PBS-CMF. Filter the solution with a 0.22 &#956;m syringe filter and store at -20 &#176;C. Note: For the enzyme digestion step of the protocol, for every set of 10 mice for extraction, 300 &#181;L of 10x collagenase dispase is required.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>31398</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>11284932001</td>
			<td>Prepare a 1000X stock solution by dissolving 100 mg in 10 ml sterile water and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G-2500</td>
			<td>Prepare the working coating by making a 0.2% gelatin solution in sterile PBS-CMF (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.86 g/L NaHPO4 (12 H2O), pH 7.4). Autoclave the solution for minimum 20 minutes at 120 &#176;C and store at room temperature. Culture dishes to be coated for at least 4 hours at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Biochrom AG</td>
			<td>A-2712</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Merck</td>
			<td>I.00289</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H-8264</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-0887</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biocoat</td>
			<td>354230</td>
			<td>Prepare a working coating solution of Matrigel by diluting stock in cold DMEM at 1:48 ratio with its final concentration to be 85 &#181;g/cm2. Cell culture dishes should be coated at least for 1 hour at room temperature.</td>
		</tr>
		<tr>
			<td>Pericyte Medium-mouse</td>
			<td>Sciencell research laboratories</td>
			<td>1231</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Tosyl Lysin Chloromethyl Ketone</td>
			<td>Sigma</td>
			<td>T-7254</td>
			<td>Prepare a 1000X stock solution in WBA by dissolving 16 mg in 10.88 mL of WBA to make a 4 mM solution and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Vitamins</td>
			<td>Sigma</td>
			<td>B-6891</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Equipment Requirements</td>
		</tr>
		<tr>
			<td>Filtration tools</td>
			<td>Sefar, Nylon mesh, 60-micron porosity</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td>Swing bucket rotor centrifuge</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath with agitator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood : BSL2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glassware (all components to be heat sterilized)</td>
			<td>Dounce Tissue Grinder With Glass Pestle</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle I: 0.0035 - 0.0065 inches</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle II: 0.0010 - 0.0030 inches</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter assembly with coarse porosity fritted glass filter support base</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical dissection tools (all components to be heat sterilized)</td>
			<td>Forceps, scissors, Bunsen burner, cotton swabs, gauge</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high output method to isolate cerebral pericytes from mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

This protocol (Figure 1) efficiently yields 9 wells (of 6-well plates) at the time of seeding at P0 (Figure 2A (P0: Day 1)).
From P0 to P2, there are specific morphological characteristics by which endothelial cells (indicated by white arrows) and s gradual increase in pericytes (indicated by black arrows) can be observed. In P0, the elongated endothelial cells developing from microvessels are in abundance (Figure 2A, P0: Day 3), while the abundance of such elongated cells is reduced in P1 and absent in P2. On the contrary, the pericytes appear as quadrilateral cells which are abundant in P2 (Figure 2A, P2: Day 18).
To confirm the purity of the pericyte culture in P2, we checked the expression of NG2, CD146 and PDGFR-beta as pericyte markers using quantitative PCR (Figure 2B), immunocytochemistry (Figure 2C), and western blot (Figure 3). Pericytes in P2 express higher levels of CD146, NG2 and PDGFR-&#946; when compared to the expression in total mouse brain (Ms Br) extract. As a control, expression of endothelial markers Occludin and CD31, astrocytes marker Glial Fibrillary Acidic Protein (GFAP) and microglia marker CD11b were also observed absent in P2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60588/60588fig01.jpg" /> 
Figure 1: Summary of the protocol. This outline represents critical steps for pericyte extraction which begins with tissue disintegration with glass pestle grinder followed by 3-step separation in dextran and filtration. This protocol employs a 33 min enzyme digestion step. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60588/60588fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60588/60588fig2a.jpg" /> 
Figure 2: Cells morphology and markers expression. (A) Phase contrast images of pericytes in P0, P1 and P2 stages of proliferation. Abundant endothelial cells in P0 are indicated by white arrows. Their number decreased in P1 and they have disappeared in P2. Pericytes are indicated by black arrows. (B) Analysis of CD146, NG2 and PDGFR-&#946; expression by PCR in pericytes in P2 with pericytes in P1 and mouse brain (Ms Br) samples. (C) Representative images of pericytes in P2 exhibiting positive immunostaining of CD146, PDGFR-&#946; and, NG2. Scale bar: 50 &#956;m (20x magnification) and 20 &#956;m (40x magnification). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60588/60588fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60588/60588fig03.jpg" /> 
Figure 3: Representative purity of the cell cultures. Analysis of CD146, PDGFR-&#946;, NG2, Occludin, GFAP, CD31, CD11b and Tubulin expression by western blotting in pericytes in P2 and mouse brain (Ms Br) samples. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60588/60588fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60588/60588fig4a.jpg" /> 
Figure 4: Representative comparison of different methods of tissue disintegration, purification, enrichment and enzymatic digestion. Each published protocol is summarized with indications on the number of animals used for each protocol. Outputs are also indicated with respect to the number of wells obtained upon seeding. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60588/60588fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High Output Method to Isolate Cerebral Pericytes from Mouse at JoVE.com

A High Output Method to Isolate Cerebral Pericytes from Mouse

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

a-high-output-method-to-isolate-cerebral-pericytes-from-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.3K Views.  University of Artois. We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Video: A High Output Method to Isolate Cerebral Pericytes from Mouse

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

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A Method for Islet Transplantation to the Omentum in Mouse