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

Yazarlar enjeksiyon için ekipman kullanımı için Dr F. Bryan Pickett ve Dr Rodney Dale teşekkür ederiz. Bu çalışma Loyola University Chicago'dan M.D., D.T., ve J.J.'e kadar araştırma fonu ile finanse edilmiştir.

1. Örneklerin Toplanması
<ol><li>Embriyo koleksiyonu
	<ol><li>Her embriyo toplama kafesinde, koleksiyonlar için 20−25 erkek ile en az 50 bakire dişi kullanın. Bu sinekleri, toplamaya başlamadan önce 1−2 gün boyunca mısır unu-agar gıdası(Malzeme Tablosu)ile bir şişede kuluçkaya yatırın23. NOT: Daha fazla sinek kullanılabilir, ancak kadın-erkek oranı 2:1 tutulmalıdır.</li>
		<li>Önceden ısıtılmış elma suyu agar t...

<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+mechanisms+in+the+Drosophila+blood-brain+barrier.">Barrier mechanisms in the Drosophila blood-brain barrier.</a> Frontiers in Neuroscience. 8, 414 (2014).</li><li>Schwabe, T., Bainton, R. J., Fetter, R. 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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurexin+IV,+caspr+and+paranodin--novel+members+of+the+neurexin+family:+encounters+of+axons+and+glia.">Neurexin IV, caspr and paranodin--novel members of the neurexin family: encounters of axons and glia.</a> Trends in Neurosciences. 21 (10), 444-449 (1998).</li><li>Baumgartner, 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=A+Drosophila+neurexin+is+required+for+septate+junction+and+blood-nerve+barrier+formation+and+function.">A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.</a> Cell. 87 (6), 1059-1068 (1996).</li><li>Banerjee, S., Pillai, A. M., Paik, R., Li, J., Bhat, M. 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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. 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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. 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Bu protokol, Geç embriyonik ve D. melanogaster gelişiminin üçüncü instar larva aşamalarında BBB bütünlüğü için test etmek için gereken adımların kapsamlı bir açıklamasını sağlar. Benzer yaklaşımlar başka bir yerde geliştirme sırasında BBB bütünlüğünü titretmek için tarif edilmiştir, yanı sıra yetişkin aşamalarında5,7,29,30. Ancak, malzeme ve y?...

Gelişim sırasında hücre-hücre iletişimi ve etkileşimleri doku ve organ yapısı nın ve fonksiyonunun kurulması açısından kritik öneme sahiptir. Bazı durumlarda, bu hücre-hücre etkileşimleri uygun organ fonksiyonu sağlamak için çevreden organları mühür. Bu kan-beyin bariyeri (BBB) tarafından izole edilir sinir sistemi için durumdur. İnsanlarda BBB disfonksiyonu epilepsi de dahil olmak üzere nörolojik bozukluklar ile bağlantılı olmuştur, ve bariyerin arıza multipl skleroz ve amiyotrofik lateral skleroz dahil nörodejeneratif hastalıklarda gözlenmiştir1. Memelilerde, BBB endotel hücreleri arasında sıkı kavşaklar oluşur2,3. Meyve sineği Drosophila melanogaster de dahil olmak üzere diğer hayvanlar, glial hücrelerden oluşan bir BBB var. Bu glial hücreler besin, atık ürünler, toksinler ve sinir sistemi içine ve dışında büyük moleküllerin hareketini kontrol etmek için seçici geçirgen bir bariyer oluşturur4. Bu hareketlilik ve koordinasyon için izin, yangın eylem potansiyelleri için gerekli elektrokimyasal degrade bakım sağlar4. D. melanogaster, glia potasyum açısından zengin, kan gibi hemolenf5sinir sistemini korumak .
Merkezi sinir sistemi (CNS) ve periferik sinir sistemi (PNS) D. melanogaster, iki dış glial tabakalar, subperineurial glia ve perineurial glia, yanı sıra ekstrasellüler matriks bir dış ağ, nöral lamella, formu hemolenf-beyin ve hemolenf-sinir bariyeri6, Bu makale boyunca BBB olarak anılacaktır. Geliştirme subperineurial glia poliploid olur ve sinir sistemi5çevreleyen büyütmek 5,6,7,8,9,10,11 . Subperineurial glia form septate kavşaklar, hemolenf ve sinir sistemi arasında ana difüzyon bariyersağlamak5,6,12. Bu kavşaklar moleküler olarak omurgalılarda miyelinating glia paranodlarında bulunan septate benzeri kavşaklar benzer, ve memelilerin BBB sıkı kavşaklar olarak aynı işlevi gerçekleştirmek13,14, 15.000 , 16.02.20 , 17. Perineurial glia bölmek, büyümek, ve metabolitleri ve büyük moleküllerin difüzyon düzenlemek için subperineurial glia etrafında sarın6,10,18,19. BBB oluşumu 25 °C5,8'deyumurtlama (AEL) sonrası 18,5 saat tamamlanır. Önceki çalışmalarda BBBoluşumununkritik düzenleyicileri olan genler tespit 20,21,22. Bu genlerin tam rollerini daha iyi anlamak için, bu potansiyel düzenleyicilerin mutasyonunun BBB bütünlüğü üzerindeki etkisini incelemek önemlidir. Önceki çalışmalarda embriyo ve larvalarda BBB bütünlüğünü nitelemek için yaklaşımlar özetlenen olsa da, bu test için kapsamlı bir protokol henüz tarif edilmemiştir5,7. Bu adım adım protokol, D. melanogaster embriyonik ve üçüncü instar larva aşamalarında BBB bütünlüğünü nitretetmek için yöntemleri açıklar.

<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

Uygun sinir sistemi gelişimi kan-beyin bariyerinin oluşumunu içerir, sıkı sinir sistemine erişimi düzenleyen ve toksinler ve patojenlerden nöral doku korur difüzyon bariyeri. Bu bariyerin oluşumundaki bozukluklar nöropatilerle ilişkilendirilmiştir ve bu bariyerin bozulması birçok nörodejeneratif hastalıkta gözlenmiştir. Bu nedenle, potansiyel terapötik hedefleri belirlemek için kan-beyin bariyerinin oluşumunu ve bakımını düzenleyen genlerin belirlenmesi önemlidir. Bu genlerin nöral gelişimde tam rollerini anlamak için, değişmiş gen ekspresyonunun kan-beyin bariyerinin bütünlüğü üzerindeki etkilerini tetkik etmek gerekir. Kan-beyin bariyerinin kurulmasında işlev moleküllerin çoğu ökaryotik türler arasında korunmuş olduğu bulunmuştur, meyve sinek de dahil olmak üzere, Drosophila melanogaster. Meyve sinekleri sinir sistemi gelişimi ve işlevini düzenleyen moleküler mekanizmaları incelemek için mükemmel bir model sistemi olduğu kanıtlanmıştır. Bu protokol, D. melanogaster gelişiminin embriyonik ve larva evrelerinde kan-beyin bariyerinin bütünlüğünü test etmek için adım adım bir prosedürü tanımlar.

Burada açıklanan yöntemler, D. melanogaster embriyoları ve larvalarında CNS boyunca BBB'nin bütünlüğünün görselleştirilmesine olanak sağlar (Şekil 1). Geç embriyogenezde BBB oluşumunun tamamlanmasından sonra, BBB fonksiyonları beyin ve VNC5büyük molekülleri dışlamak için . Bu protokol BBB oluşumunu test etmek için bu işlevden yararlanır. Yabani tip (Oregon R) geç evre 17 (20−21 saat eski) embriyolara sulforhodamine 101 asit klo...

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

Kan-beyin bariyerbütünlüğü sinir sistemi fonksiyonu için çok önemlidir. Drosophila melanogasterolarak, kan-beyin bariyeri geç embriyogenez sırasında glial hücreler tarafından oluşur. Bu protokol, D. melanogaster embriyolarında ve üçüncü instar larvalarında kan-beyin bariyeri oluşumu ve bakımı için taht yöntemlerini açıklamaktadır.

Drosophila melanogaster Kan-beyin Bariyer Bütünlüğü için Tahse

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

Bu yöntem drosophila gelişiminin birden fazla aşamasında kan-beyin bariyerinin oluşumu ve bakımı genetik düzenleme ile ilgili anahtar sorulara cevap yardımcı olabilir. Bu yöntem aynı zamanda çeşitli organizmalarda bağırsak epiteli gibi diğer bariyerlerin geçirgenliğini incelemek için uyarlanabilir. Bu tekniğin en büyük avantajı canlı dokularda kan-beyin bariyer fonksiyonunun değerlendirilmesi sağlar olmasıdır.Örnek manipülasyonlar zor olabilir ve birçok adım ayrıntılara yakın dikkat gerektirdiğinden, bu yöntemin görsel gösterimi çok önemlidir. Embriyo toplama için Mısır Unu Agar gıda ile şişe başına 20-25 erkek ile en az 50 bakire kadın yer ve koleksiyonları başlamadan önce bir ila iki gün boyunca bu sinekler kuluçka. Toplama bir gün önce, 25 santigrat derece gece sıcak elma suyu agar plakaları.Ertesi sabah transfer karbondioksit anestezi sinekler bir toplama kafesi ve kafesin açık ucunda maya ezmesi küçük bir yayma ile önceden ısıtılmış elma suyu agar plaka yerleştirin. Sonra kırmızı kollu kafese plaka güvenli. Eski embriyoları temizlemek için, sinekler 25 santigrat derece bir saat boyunca bir elma suyu agar plaka üzerinde yumurta bırakmak için izin verin.Kuluçka sonunda, kafes örgü tarafı aşağı ters ve kafesin altına sinek dokunun. Maya hamuru küçük bir yayma ile yeni bir önceden ısıtılmış elma suyu agar plaka ile elma suyu agar değiştirin. Sinekler 25 santigrat derece başka bir saat için yeni plaka üzerinde yumurtlamak için izin verin.Enjeksiyon için geç evre 17 embriyoları toplamak için, maya ezmesi bir yayma ile yeni bir önceden ısıtılmış plaka ile elma suyu agar plaka değiştirin. Ve sineklerin 25 santigrat derecede yumurtalarını tabağa bırakmalarına izin verin. Bir saat sonra, plakayı değiştirin ve plakayı 25 santigrat derecede 19 saat boyunca toplanan embriyolarla değiştirin, böylece embriyolar görüntüleme sırasında 20 ila 21 saat yaşlansın.19 saatlik kuluçka sonunda, embriyo toplama plakasını kuvözden çıkarın ve plakanın yüzeyini PbTx ile kapatın. Ve embriyolar ile süzgeç yerleştirin 50%çamaşır suyu çözeltisi içinde 100 milimetre Petri çanak beş dakika oda sıcaklığında zaman zaman ajitasyon ile onları dekorionate.Kuluçka sonunda, her yıkama için yeni bir Petri kabı kullanarak, taze PbTx süzgeç girdap tarafından üç kez embriyolar durulayın. PbTx ile süzgeç bir tarafında yıkama embriyolar ve% 2 agarose jel levha üzerine embriyolar aktarmak için bir cam pipet kullanın. Fazla sıvıyı filtre kağıdı yla çıkarın.Altı ila sekiz embriyoyu levhaüzerinde, sağdaki arka larla hizalayın ve sırt kenarları yukarı bakacak şekilde ve embriyoların üzerine yapıştırılmış çift taraflı bant parçasına sıkıca basın. Daha sonra örnekleri Halokarbon yağı ile kaplamadan önce embriyoları oda sıcaklığında yaklaşık 25 dakika kurutun. Larva koleksiyonu için, istenilen genotip 5-10 bakire dişi sinekler ve Cornmeal Agar gıda ile bir şişe istenilen genotip yarısı kadar erkek ile bir haç kurmak.25 santigrat derece de beş ila yedi gün sonra, genotip bağlı olarak, yavaşça şişe den dolaşan üçüncü yıldız larvaları toplamak ve herhangi bir sıkışmış gıda kaldırmak için PBS ile larvaları durulayın için forceps kullanın. Larvaları bir doku üzerinde kurutmak için bir boya fırçası kullanmadan önce gerektiği gibi genotipleme için bir elma suyu agar plakasına aktarın. Daha sonra altı ila sekiz larvayı çift taraflı bantla hazırlanmış bir slayta aktarmak için forcep kullanın.Enjeksiyondan önce, d.Melanogaster enjeksiyonları için standart bir iğne şekli içine kılcal tüpler çekmek ve bir Petri kabında kil iğneleri çapa bir mikropipet puller kullanın. Embriyo enjeksiyonu için, 20 mikrolitrejel yükleme pipet ucu sülforhodamine 101 asit klorür konjuge 10 kilodalton dekstran 5 mikrolitre ile hazırlanmış bir iğne yüklemek ve bir iğne tutucu içine iğne yüklemek kullanın. İğneyi çelik bir tabana sabitlenmiş bir mikromanipülatöre yerleştirin ve enjekte cihazını inç kare başına 50 pound'a ve 10'luk bir aralıkla 5 ila 10 milisaniyeye ayarlayın.Mikromanipülatör aşamasında slayt yerleştirin ve 45 derecelik bir açıyla çift taraflı bant kenarına iğne kenarına fırça sadece açılış yoluyla 10 kilodalton dextran akışını sağlamak için yeterli kırık hafif açılı bir ucu oluşturmak için. Boya iğnenin ucunda olana kadar ayak pedalını pompalayın. İğneyi embriyoya paralel olacak şekilde hizala ve numunenin arka ucunu delin.Numunenin içine iki nanolitre boya enjekte etmek için ayak pedalını pompalayın. Kuluçka amacıyla enjeksiyon süresine dikkat edin ve ek numuneler enjekte etmek için slaytaşağı devam edin. Larva enjeksiyonu için, embriyo enjekte etmek için kullanılandan biraz daha büyük açılı bir açıklık yapın ve larvalara embriyonik örneklerde gösterildiği gibi 220 nanolitre enjekte etmeden önce iğneyi numuneye doğru hafifçe aşağıya doğru açıya çevirin.10 dakikalık enjeksiyon kuluçka sonunda, bir spacer olarak slayt örneklerinin sağ ve sol taraflarında petrol jöle uygulamak için bir pamuk uçlu aplikatör kullanın. Kapak fişini üste yerleştirin ve kaydırağı konfokal mikroskop aşamasına yerleştirin. 20X hedefini seçin ve her embriyonun derinliği boyunca örnekleri görüntüleyin.Daha sonra formüle göre kan-beyin bariyeri tehlikeye ile örneklerin yüzdesini hesaplayın. Diseksiyon işlemine başlamadan önce, bir slaytüzerine yaklaşık 0,5 santimetre aralıklı iki kapak fişi takmak için oje kullanın ve 30 dakikalık kuluçka sonunda slaytta PBS içine enjekte edilen larva yerleştirin. Bir çift forceps kullanarak, larvayı yarım kavrayışla kavrayın ve larvanın ön ve arka yarısını ayırmak için ikinci bir çift forceps kullanın.Daha sonra, ağız kancalarında ön bölgeyi kavramak için bir çift forceps kullanın ve ilk çift forceps ucu üzerinde vücut duvarını tersine çevirmek için ikinci çift forceps kullanın. Beyin ve ventral sinir kordonu açığa çıkacak. Gerekirse sinirleri keserek beyin ve ventral sinir kordonu vücut duvarından ayırın ve slayt vücut duvarı kaldırın.% 80 gliserol 10 mikrolitre ile örnek kapağı. Daha sonra görüntüleme ve görüntü örnekleri için örnek üstüne bir kapak kayma yerleştirin kanıtgösterildiği gibi bir tehlikeye kan-beyin bariyeri ile örneklerin yüzdesini belirlemek için. Yabani tip geç evre 17 embriyolar 10 kilodalton dekstran ile sülforhodamine 101 asit klorür floresan boya konjuge enjekte edildiğinde, büyük dekstran molekülü ventral sinir kordonu dışında tutulur, beklendiği gibi.Heterozigot ham 1 mutant embriyolar sağlam bir kan-beyin bariyeri sergilerler, vahşi tip kontrol embriyolarında gözlenene benzer. Buna karşılık, homozigot ham 1 mutant embriyolar kan-beyin bariyerinin bütünlüğü kusurları sergilemek, ile 10 kilodalton dextran ventral sinir kordonu içine sel, oluşturmak için kan-beyin bariyerinin bir başarısızlık gösteren. Yabani tip kontrol larva örneklerinde, 10 kilodalton dextran kan-beyin bariyerini geçemez ve beyin ve ventral sinir kordonu dışında tutulur.Bu prosedür denerken, embriyoların düzgün yaşlanması, örneklerin kan-beyin bariyerinin tam laşmasının beklendiği bir yaşta incelenmesini sağlamak için önemlidir. Bu prosedürütaki kan-beyin bariyerini koruyan mutantlar, hücresel düzeydeki gen fonksiyonunu daha iyi anlamak için genetik, immünohistokimya ve mikroskopi kullanılarak daha da ileri ye doğru çalışılabilirler. Bu teknik, araştırmacıların kan-beyin bariyerinin kurulması ve bakımı için kritik olan ek genleri belirlemelerine olanak sağlayacaktır.

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.

Ve tahlil Kan Hücresi popülasyonlar<em&gt; Drosophila melanogaster</em&gt; Larva

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

Gelişim Biyolojisi

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.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

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.

Meyve Fly Yetiştirme<em&gt; Drosophila melanogaster</em&gt; Aksenik ve gnotobiyotik Koşullarında

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.

Sineği boyanmasına ile steroidogenik organları ve İnteraktif Organlar görselleştirme için Protokoller<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.

Diseksiyon ve mantar vücut immünfloresan boyama ve yetişkin Drosophila melanogaster beynindeki nöronlar Photoreceptor

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.

Fare kan-Testis bariyeri bütünlük çalışma için In Vivo yöntemi

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.

Drosophila melanogaster endokrin bozulma test yöntemleri

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.

İnsan iPSC Tabanlı Kan-Beyin Bariyer Chip Üretimi

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.

Mikro bilgisayarlı Tomografi kullanılarak Drosophila melanogaster'ın Tüm Hayvan Görüntülemesi

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

Drosophila melanogaster Üçüncü Instar Larvalarından Canlı Beyin Eksplantlarının Görüntülenmesi

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.

Yazar Gündemi: Asimetrik Hücre Bölünmesi Dinamiğinin Araştırılması: Drosophila Larva Beyin Eksplantlarının Canlı Görüntülenmesi İçin Bir Protokol 

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.

Drosophila melanogaster Üçüncü Instar Larva Beyinlerinin Canlı Hücre Görüntülemesi

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