Name: assay for blood-brain barrier integrity in drosophila melanogaster
Uploaded: 2019-09-18T15:00:00
Description: Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Författarna tackar Dr. F. Bryan Pickett och Dr Rodney Dale för användning av utrustning för injektion. Detta arbete finansierades genom forskningsfinansiering från Loyola University Chicago till MD, D.T., och J.J.

1. provtagning
<ol><li>Embryosamling
	<ol><li>I varje embryosamlingsbur, Använd minst 50 jungfru honor med 20 − 25 hanar för samlingar. Inkubera dessa flugor i en flaska med majsmjöl-agar mat (tabell över material) för 1 − 2 dagar före början samlingar23. Anmärkning: Fler flugor kan användas, men förhållandet mellan kvinnor och män bör hållas vid 2:1.</li>
		<li>Pre-Warm äppeljuice agar tallrikar (tabell 1)...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctions+between+intimately+apposed+cell+membranes+in+the+vertebrate+brain.">Junctions between intimately apposed cell membranes in the vertebrate brain.</a> Journal of Cell Biology. 40 (3), 648-677 (1969).</li><li>Tietz, S., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers:+Crosstalk+between+complex+tight+junctions+and+adherens+junctions.">Brain barriers: Crosstalk between complex tight junctions and adherens junctions.</a> Journal of Cell Biology. 209 (4), 493-506 (2015).</li><li>Hindle, S. J., Bainton, R. 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D., Heberlein, U., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+signaling+is+required+for+blood-brain+barrier+formation+in+drosophila.">GPCR signaling is required for blood-brain barrier formation in drosophila.</a> Cell. 123 (1), 133-144 (2005).</li><li>Stork, 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=Organization+and+function+of+the+blood-brain+barrier+in+Drosophila.">Organization and function of the blood-brain barrier in Drosophila.</a> Journal of Neuroscience. 28 (3), 587-597 (2008).</li><li>Unhavaithaya, Y., Orr-Weaver, T. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+cell+cycles+regulated+by+Notch+signaling+control+cell+size+and+ensure+a+functional+blood-brain+barrier.">Variant cell cycles regulated by Notch signaling control cell size and ensure a functional blood-brain barrier.</a> Development. 145 (3), dev157115 (2018).</li><li>von Hilchen, C. M., Beckervordersandforth, R. M., Rickert, C., Technau, G. M., Altenhein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity,+origin,+and+migration+of+peripheral+glial+cells+in+the+Drosophila+embryo.">Identity, origin, and migration of peripheral glial cells in the Drosophila embryo.</a> Mechanisms of Development. 125 (3-4), 337-352 (2008).</li><li>Beckervordersandforth, R. M., Rickert, C., Altenhein, B., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtypes+of+glial+cells+in+the+Drosophila+embryonic+ventral+nerve+cord+as+related+to+lineage+and+gene+expression.">Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression.</a> Mechanisms of Development. 125 (5-6), 542-557 (2008).</li><li>Bellen, H. J., Lu, Y., Beckstead, R., Bhat, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+ensheathment+and+septate+junction+formation+in+the+peripheral+nervous+system+of+Drosophila.">Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila.</a> Journal of Neuroscience. 26 (12), 3319-3329 (2006).</li><li>Bhat, M. 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=Axon-glia+interactions+and+the+domain+organization+of+myelinated+axons+requires+neurexin+IV/Caspr/Paranodin.">Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.</a> Neuron. 30 (2), 369-383 (2001).</li><li>Faivre-Sarrailh, 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=Drosophila+contactin,+a+homolog+of+vertebrate+contactin,+is+required+for+septate+junction+organization+and+paracellular+barrier+function.">Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function.</a> Development. 131 (20), 4931-4942 (2004).</li><li>Salzer, J. L., Brophy, P. J., Peles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+domains+of+myelinated+axons+in+the+peripheral+nervous+system.">Molecular domains of myelinated axons in the peripheral nervous system.</a> Glia. 56 (14), 1532-1540 (2008).</li><li>von Hilchen, C. M., Bustos, A. E., Giangrande, A., Technau, G. M., Altenhein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predetermined+embryonic+glial+cells+form+the+distinct+glial+sheaths+of+the+Drosophila+peripheral+nervous+system.">Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system.</a> Development. 140 (17), 3657-3668 (2013).</li><li>Matzat, 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=Axonal+wrapping+in+the+Drosophila+PNS+is+controlled+by+glia-derived+neuregulin+homolog+Vein.">Axonal wrapping in the Drosophila PNS is controlled by glia-derived neuregulin homolog Vein.</a> Development. 142 (7), 1336-1345 (2015).</li><li>Limmer, S., Weiler, A., Volkenhoff, A., Babatz, F., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+blood-brain+barrier:+development+and+function+of+a+glial+endothelium.">The Drosophila blood-brain barrier: development and function of a glial endothelium.</a> Frontiers in Neuroscience. 8, 365 (2014).</li><li>Ho, T. 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=Expressional+Profiling+of+Carpet+Glia+in+the+Developing+Drosophila+Eye+Reveals+Its+Molecular+Signature+of+Morphology+Regulators.">Expressional Profiling of Carpet Glia in the Developing Drosophila Eye Reveals Its Molecular Signature of Morphology Regulators.</a> Frontiers in Neuroscience. 13, 244 (2019).</li><li>DeSalvo, M. K., 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+Drosophila+surface+glia+transcriptome:+evolutionary+conserved+blood-brain+barrier+processes.">The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes.</a> Frontiers in Neuroscience. 8, 346 (2014).</li><li>. BDSC Cornmeal Food Available from: <a target="_blank" href="https://bdsc.indiana.edu/information/recipes/bloomfood.html">https://bdsc.indiana.edu/information/recipes/bloomfood.html</a> (2017)</li><li>Le, 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=A+new+family+of+Drosophila+balancer+chromosomes+with+a+w-+dfd-GMR+yellow+fluorescent+protein+marker.">A new family of Drosophila balancer chromosomes with a w- dfd-GMR yellow fluorescent protein marker.</a> Genetics. 174 (4), 2255-2257 (2006).</li><li>Casso, D., Ramirez-Weber, F. A., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-tagged+balancer+chromosomes+for+Drosophila+melanogaster.">GFP-tagged balancer chromosomes for Drosophila melanogaster.</a> Mechanisms of Development. 88 (2), 229-232 (1999).</li><li>Halfon, M. 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=New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes.">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34 (1-2), 135-138 (2002).</li><li>Miller, D. F., Holtzman, S. L., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customized+microinjection+glass+capillary+needles+for+P-element+transformations+in+Drosophila+melanogaster.">Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster.</a> BioTechniques. 33 (2), 366-372 (2002).</li><li>Luong, D., Perez, L., Jemc, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+raw+as+a+regulator+of+glial+development.">Identification of raw as a regulator of glial development.</a> PLoS One. 13 (5), e0198161 (2018).</li><li>Pinsonneault, R. L., Mayer, N., Mayer, F., Tegegn, N., Bainton, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+models+for+studying+the+blood-brain+and+blood-eye+barriers+in+Drosophila.">Novel models for studying the blood-brain and blood-eye barriers in Drosophila.</a> Methods in Molecular Biology. 686, 357-369 (2011).</li><li>Love, C. R., Dauwalder, B., Barichello, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+Model+to+Study+the+Blood-Brain+Barrier.">Drosophila as a Model to Study the Blood-Brain Barrier.</a> Blood-Brain Barrier. , 175-185 (2019).</li><li>Lin, D. M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+and+increased+expression+of+Fasciclin+II+alters+motoneuron+growth+cone+guidance.">Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance.</a> Neuron. 13 (3), 507-523 (1994).</li><li>Sepp, K. J., Schulte, J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+glia+direct+axon+guidance+across+the+CNS/PNS+transition+zone.">Peripheral glia direct axon guidance across the CNS/PNS transition zone.</a> Developmental Biology. 238 (1), 47-63 (2001).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Devraj, K., Guerit, S., Macas, J., Reiss, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vivo+Blood-brain+Barrier+Permeability+Assay+in+Mice+Using+Fluorescently+Labeled+Tracers.">An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.</a> Journal of Visualized Experiments. 132, e57038 (2018).</li><li>Fairchild, M. J., Smendziuk, C. 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Detta protokoll ger en omfattande beskrivning av de steg som behövs för att assay för BBB integritet under sena embryonala och tredje INSTAR larvstadier av D. melanogaster utveckling. Liknande metoder har beskrivits på andra ställen för att assay integriteten av BBB under utveckling, samt i vuxen stadier5,7,29,30. Beskrivningar av procedurer i material-och metod avsnitt är dock ...

Under utveckling, cell-cellkommunikation och interaktioner är avgörande för inrättandet av vävnad och organ struktur och funktion. I vissa fall, dessa cell-cell interaktioner tätning av organ från den omgivande miljön för att säkerställa korrekt organfunktion. Detta är fallet för nervsystemet, som isoleras av blod-hjärnbarriären (BBB). Dysfunktion av BBB hos människor har kopplats till neurologiska sjukdomar inklusive epilepsi, och nedbrytning av barriären har observerats i neurodegenerativa sjukdomar inklusive multipel skleros och amyotrofisk lateral skleros1. Hos däggdjur bildas BBB genom täta korsningar mellan endotelceller2,3. Andra djur, inklusive bananfluga, Drosophila melanogaster, har en BBB består av gliaceller. Dessa gliaceller celler bildar en selektivt permeabel barriär för att kontrollera rörligheten för näringsämnen, slaggprodukter, toxiner, och stora molekyler in i och ut ur nervsystemet4. Detta möjliggör underhåll av den elektrokemiska lutning som krävs för att avfyra actionpotentialer, möjliggör rörlighet och samordning4. I D. melanogasterskyddar glia nervsystemet från den kalium rika, blodliknande hemolymfa5.
I centralanervsystemet (CNS) och perifera nervsystemet (PNS) av D. melanogaster, två yttre glialskikt, subperineurial glia och perineurial glia, samt ett yttre nätverk av extracellulär matris, den neurala lamellerna, bildar den hemolymfa-hjärna och hemolymfa-nerv barriär6, kallad BBB hela denna artikel. Under utveckling subperineurial glia bli polyploida och förstora för att omge nervsystemet5,6,7,8,9,10,11 . Subperineurial glia bildar septate korsningar, som ger den huvudsakliga diffusion barriären mellan hemolymfa och nervsystemet5,6,12. Dessa korsningar är molekylärt liknar septate-liknande korsningar finns på paranodes av myelinating glia i ryggradsdjur, och de utför samma funktion som täta korsningar i BBB av däggdjur13,14, 15 , 16 , 17. den perineurial glia dividera, växa, och Linda runt subperineurial glia att reglera spridningen av metaboliter och stora molekyler6,10,18,19. BBB-bildningen är avslutad med 18,5 h efter äggläggningen (AEL) vid 25 ° c5,8. Tidigare studier har identifierat gener som är kritiska regulatorer av BBB-formation20,21,22. För att bättre förstå de exakta rollerna för dessa gener, är det viktigt att undersöka effekten av mutation av dessa potentiella tillsynsmyndigheter på BBB integritet. Medan tidigare studier har skisserat metoder för testmetoder BBB integritet i embryon och larver, ett omfattande protokoll för denna analys har ännu inte beskrivits5,7. Detta steg-för-steg-protokoll beskriver metoder för testmetoder BBB integritet under D. melanogaster embryonala och tredje INSTAR larvstadier.

<table>
	<tbody>
		<tr>
			<td>10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran</td>
			<td>ThermoFisher Scientific</td>
			<td>D1863</td>
			<td>Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL.</td>
		</tr>
		<tr>
			<td>20 &#956;L Gel-Loading Pipette Tips</td>
			<td>Eppendorf</td>
			<td>22351656</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol (200 proof)</td>
			<td>Pharmco-Aaper</td>
			<td>11000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Active Dry Yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>DeWalt</td>
			<td>D55140</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple Juice</td>
			<td>Mott&#39;s Natural Apple Juice</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Household Bleach</td>
			<td></td>
			<td>1-5% Hypochlorite</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-277</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Samples imaged using 20x objective (UPlanSApo 20x/ 0.75)</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Applicator</td>
			<td>Puritan</td>
			<td>19-062614</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape 1/2&#34;</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers; Pattern #5; .05 X .01mm Tip</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Bottles</td>
			<td>Fisher Scientific</td>
			<td>AS-355</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Vials</td>
			<td>Fisher Scientific</td>
			<td>AS-515</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot Pedal</td>
			<td>Treadlite II</td>
			<td>T-91-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Caster</td>
			<td>Bio-Rad</td>
			<td>1704422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Tray</td>
			<td>Bio-Rad</td>
			<td>1704436</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated sugar</td>
			<td></td>
			<td></td>
			<td>Purchased from grocery store.</td>
		</tr>
		<tr>
			<td>Halocarbon Oil</td>
			<td>Lab Scientific, Inc.</td>
			<td>FLY-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Schott</td>
			<td>Ace I</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator Stand</td>
			<td>World Precision Instruments</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>Nightsea Filter Sets</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-LFS-CY</td>
			<td>For visualization of YFP</td>
		</tr>
		<tr>
			<td>Nightsea Full Adapter System w/ Royal Blue Color Light Head</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-RB</td>
			<td>For visualization of GFP</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Simply Simmons</td>
			<td>Chisel Blender #6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter</td>
			<td>Fisher Scientific</td>
			<td>13-683C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Pump</td>
			<td>World Precision Instruments</td>
			<td>PV830</td>
			<td>This is also referred to as a microinjector or pressure regulator. Since the model used in our study is no longer available this is one alternative.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Embryo Collection Cages</td>
			<td>Genesee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Base Plate</td>
			<td>World Precision Instruments</td>
			<td>5052</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000</td>
			<td>Used for tissue dissection.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with transmitted light source</td>
			<td>Baytronix</td>
			<td></td>
			<td>Used for injection.</td>
		</tr>
		<tr>
			<td>Tegosept (p-hydroxybenzoic acid, methyl ester)</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Vial Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-273</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay for blood-brain barrier integrity in drosophila melanogaster

Korrekt nervsystemet utveckling omfattar bildandet av den Blood-brain barriären, diffusions barriären som tätt reglerar tillgång till nervsystemet och skyddar nervvävnad från toxiner och patogener. Defekter i bildandet av denna barriär har korrelerats med neuropatier, och nedbrytningen av denna barriär har observerats i många neurodegenerativa sjukdomar. Därför är det viktigt att identifiera de gener som reglerar bildandet och underhållet av blod-hjärnbarriären för att identifiera potentiella terapeutiska mål. För att förstå de exakta rollerna dessa gener spelar i neurala utveckling, är det nödvändigt att analysen effekterna av förändrade genuttryck på integriteten av blod-hjärnbarriären. Många av de molekyler som fungerar i upprättandet av blod-hjärnbarriären har visat sig vara bevaras över eukaryota arter, inklusive bananfluga, Drosophila melanogaster. Fruktflugor har visat sig vara ett utmärkt modellsystem för att undersöka de molekylära mekanismerna som reglerar nervsystemets utveckling och funktion. Detta protokoll beskriver ett steg-för-steg-förfarande för att assay för blod-hjärnbarriären integritet under embryonala och larvstadier av D. melanogaster utveckling.

De metoder som beskrivs här möjliggör visualisering av integriteten hos BBB i hela CNS i D. melanogaster embryon och larver (figur 1). Efter avslutad BBB formation i sena embryogenes, BBB funktioner för att utesluta stora molekyler från hjärnan och VNC5. Detta protokoll utnyttjar denna funktion för att assay BBB formation. När Wild-Type (Oregon R) sent stadium 17 (20 − 21 h gamla) embryon injicerades med 10 kDa dextran konjugerat till sulforodamin 1...

Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blod-hjärnbarriären integritet är avgörande för nervsystemets funktion. I Drosophila melanogasterbildas blod-hjärnbarriären av glialceller under sen embryogenes. Detta protokoll beskriver metoder för att analys för blod-hjärnbarriären bildning och underhåll i D. melanogaster embryon och tredje INSTAR larver.

Analys för blod-hjärnbarriären integritet i Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Denna metod kan hjälpa till att besvara viktiga frågor angående den genetiska regleringen av bildning och underhåll av blod - hjärnbarriären under flera stadier av drosophila utveckling. Denna metod kan också anpassas för att undersöka genomsläppligheten av andra hinder, som tarmepitelet i en mängd olika organismer. Den största fördelen med denna teknik är att det möjliggör utvärdering av blod - hjärnbarriären funktion i levande vävnader.Visuell demonstration av denna metod är kritisk, eftersom provmanipulationerna kan vara svåra och många steg kräver stor uppmärksamhet på detaljer. För embryosamling placera minst 50 jungfruliga honor med 20 till 25 hanar per flaska med Corn Meal Agar mat och ruva dessa flugor i en till två dagar innan du börjar samlingarna. Dagen före kollektionen, varm äppeljuice agar tallrikar på 25 grader Celsius över natten.Nästa morgon överföra koldioxid sövda flugor till en samling bur och placera en förvmat äppeljuice agar tallrik med ett litet utstryk av jästpasta på den öppna änden av buren. Sedan säkra plattan till buren med den röda hylsan. För att rensa äldre embryon, låt flugorna lägga ägg på en äppeljuice agar tallrik i en timme vid 25 grader Celsius.I slutet av inkubationen, invertera burnätsidan nedåt och knacka på flugorna till botten av buren. Byt ut äppeljuiceagarn mot en ny förvmat äppeljuiceagarplatta med ett litet utstryk av jästpasta. Låt flugorna lägga ägg på den nya plattan i ytterligare en timme vid 25 grader Celsius.För att samla sent stadium 17 embryon för injektion, ersätta äppeljuice agar plattan med en ny förvmat plattan med ett utstryk av jäst pasta. Och låt flugorna lägga ägg på tallriken vid 25 grader Celsius. Efter en timme, byt ut plattan och ålder plattan med insamlade embryon i 19 timmar vid 25 grader Celsius så att embryona kommer att vara 20 till 21 timmar gamla vid tidpunkten för avbildning.I slutet av den 19 timmars inkubation, ta bort embryot samlingsplattan från inkubatorn och täck ytan av plattan med PbTx. Använd en pensel för att lossa embryon från plattan ytan till en 70 mikrometer nylon maska sil. Och placera silen med embryon i 50% blekmedel lösning i en 100 millimeter Petriskål i fem minuter med tillfällig agitation vid rumstemperatur för att demörda dem.I slutet av inkubationen sköljer du embryona tre gånger genom att virvla silen i färsk PbTx, med hjälp av en ny petriskål för varje tvätt. Tvätta embryon på ena sidan av silen med PbTx och använd en glaspipett för att överföra embryona till en 2%agarosgelplatta. Ta bort överflödig vätska med filterpapper.Justera sex till åtta embryon på plattan med de bakre till höger och ryggsidorna uppåt och tryck fast en bit dubbelsidig tejp fäst på en bild ovanpå embryona. Därefter utse embryona i rumstemperatur i ca 25 minuter innan du täcker proverna med Halocarbon olja. För larvsamling, ställ in ett kors med 5 till 10 jungfruliga kvinnliga flugor av önskad genotyp och hälften så många män av önskad genotyp i en injektionsflaska med Cornmeal Agar-mat.Efter fem till sju dagar vid 25 grader Celsius, beroende på genotyp, använd forceps för att försiktigt samla vandrande tredje instar larver från injektionsflaskan och skölj larverna med PBS för att avlägsna eventuell fast mat. Överför larverna till en äppeljuice agarplatta för genotypning vid behov innan du använder en pensel för att torka larverna på en vävnad. Använd sedan tes för att överföra sex till åtta larver till en bild som är förberedd med dubbelsidig tejp.Före injektionen, använd en mikropipette puller för att dra kapillärrör i en standard nål form för D.Melanogaster injektioner och förankra nålarna i lera i en Petri-skålen. För embryoinjektion, använd en 20 mikroliter gel-loading pipett spets för att ladda en förberedd nål med 5 mikroliter på 10 kilodalton dextran konjugerat till sulforhodamine 101 syraklorid och ladda nålen i en nål hållare. Placera nålen i en micromanipulator säkrad till en stålbas och ställ in injektionsapparaten till 50 pounds per kvadrattum och 5 till 10 millisekunder med en räckvidd på 10.Placera bilden på mikromanipulator scenen och borsta kanten av nålen mot kanten av den dubbelsidiga tejpen i en 45 graders vinkel för att skapa en något vinklad spets bruten precis tillräckligt för att tillåta flöde av 10 kilodalton dextran genom öppningen. Pumpa fotpedalen tills färgämnet är vid nålspetsen. Rikta nålen så att den är parallell med embryot och punktera den bakre änden av preparatet.Pumpa fotpedalen för att injicera två nanoliter av färgämne i preparatet. Observera injektionstiden för inkubationsändamål och fortsätt nedåt i objektglaset för att injicera ytterligare preparat. För larvinjektion, gör en något större vinklad öppning än den som används för att injicera embryon och vinkla nålen något nedåt mot provexemplar innan du injicerar larverna med 220 nanoliter som liknar det som visas för embryonala exemplar.I slutet av inkuberingen på 10 minuters injektion använder du en bomullspetsad applikator för att applicera petroleumjelly på provens högra och vänstra sida på rutschkanan som distans. Placera luckan ovanpå och placera bilden på konfokalmikroskopets stadium. Välj 20X-målsättningen och avbilda proverna under hela djupet av varje embryo.Beräkna sedan andelen prover med en komprometterad blod - hjärnbarriären enligt formeln. Innan du börjar dissekeringsproceduren, använd nagellack för att montera två täckslilar som är fördelade ungefär 0,5 centimeter från varandra på en glidbana och placera en injicerad larva i PBS på glid i slutet av inkuberingen på 30 minuter. Med hjälp av ett par tåtor, fatta larven halvvägs ner larvkroppen och använd ett andra par tåtor för att separera larvens främre och bakre halvor.Använd därefter ett par tåtor för att greppa den främre regionen vid munhakarna och använd det andra paren av tövövingar för att invertera kroppsväggen över spetsen på det första paren av tövövingar. Hjärnan och ventral nervsladd kommer att exponeras. Separera hjärnan och ventral nervsladd från kroppen väggen genom att bryta nerver om det behövs och ta bort kroppen väggen från bilden.Täck provet med 10 mikroliter av 80%glycerol. Placera sedan en täckslirning ovanpå provet för avbildning och avbilda proverna för att bestämma andelen prover med en komprometterad blod - hjärnbarriär som visats. När vilda typ sent stadium 17 embryon injiceras med 10 kilodalton dextran konjugerad till sulforhodamine 101 syraklorid fluorescerande färgämne, den stora dextran molekylen är utesluten från den ventrala nervsladd, som förväntat.Heterozygous rå 1 mutanta embryon uppvisar en intakt blod - hjärnbarriären, liknande den som observerats i vilda typ kontroll embryon. Däremot uppvisar homozygous rå 1 mutanta embryon defekter i integriteten hos blod - hjärnbarriären, med 10 kilodalton dextran översvämningar i ventrala nerv sladden, vilket tyder på ett misslyckande av blod - hjärnbarriären att bilda. I vild typ kontroll larvprover, 10 kilodalton dextran misslyckas med att tränga in i blod - hjärnbarriären och är utesluten från hjärnan och ventral nerv sladd.Vid försök till detta förfarande är korrekt åldrande av embryona avgörande för att säkerställa att proverna undersöks vid en ålder då blod - hjärnbarriärbildning förväntas vara fullständig. Efter detta förfarande, mutanter uppvisar en komprometterad blod - hjärnbarriären kan ytterligare studeras med hjälp av genetik, immunohistokemi och mikroskopi för att bättre förstå genfunktion på cellnivå. Denna teknik gör det möjligt för forskare att identifiera ytterligare gener som är avgörande för inrättandet och upprätthållandet av blod - hjärnbarriären.

assay-for-blood-brain-barrier-integrity-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

Analysera blodcellpopulationer av<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analys av Zebrafish Larver skelettmuskulatur Integrity med Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

Uppfödning bananfluga<em&gt; Drosophila melanogaster</em&gt; Under axenisk och gnotobiotiska Villkor

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Protokoll för Visualisering steroidogena organ och deras interaktiva organ med immunfärgning i fruktflugan<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissektion och Immunofluorescerande färgning av svamp kropp och ljusmätare nervceller i vuxen Drosophila melanogaster hjärnor

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

En In Vivo -Metod för att studera mus blod-Testis barriär integritet

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Metoder för att testa endokrina störningar i Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generation av en mänsklig iPSC-baserade Blod-Brain Barriär Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Hel djuravbildning av Drosophila melanogaster med mikrodatortomografi

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Avbildning av levande hjärnexplantat från Drosophila melanogaster Third Instar-larver

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Författare Spotlight: Undersöker asymmetrisk celldelningsdynamik: Ett protokoll för live-avbildning av Drosophila-larvhjärnexplantat 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Levande cellavbildning av Drosophila melanogaster Tredje Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...