Name: long-term in vivo tracking of inflammatory cell dynamics within drosophila pupae
Uploaded: 2018-06-14T14:45:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae at JoVE.com

हम उपयोगी चर्चा के लिए मार्टिन, Nobes, रिचर्डसन और लकड़ी प्रयोगशालाओं के सदस्यों को धंयवाद देना चाहूंगा । हम भी Wolfson (ब्रिस्टल, ब्रिटेन के विश्वविद्यालय), ब्लूमिंगटन स्टॉक सेंटर (इंडियाना विश्वविद्यालय, यूएसए) और वियना Drosophila संसाधन केंद्र ( Drosophila स्टॉक्स के लिए) और Flybase (अप करने के लिए तारीख के लिए Drosophila धंयवाद जीन एनोटेशन) । इस काम के लिए एक MRC परियोजना अनुदान द्वारा p.m. और W.W. (MR/J002577/1), W.W. के लिए एक वेलकम ट्रस्ट वरिष्ठ फैलोशिप और एक वेलकम ट्रस्ट अन्वेषक पुरस्कार द्वारा समर्थन किया गया था ।

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blood-borne+PDGF/VEGF-like+ligand+initiates+wound-induced+epidermal+cell+migration+in+Drosophila+larvae.">A blood-borne PDGF/VEGF-like ligand initiates wound-induced epidermal cell migration in Drosophila larvae.</a> Current Biology. 19 (17), 1473-1477 (2009).</li><li>Burra, S., Wang, Y., Brock, A. R., Galko, M. 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लेखकों ने घोषणा की कि वे हितों का कोई प्रतिस्पर्धी संघर्ष नहीं है.

<table>
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged E-cadherin 
			;Ubi-p63E-shg.GFP; (chrII)</td>
			<td>Kyoto Stock Center, DGRC</td>
			<td>#109007</td>
			<td>Ubi-p63E promoter sequences drive expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged E-cadherin 
			;;Ubi-p63E-shg.GFP (III)</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#58742</td>
			<td>Ubi-p63E promoter sequences drive expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged Moesin 
			P{sGMCA}3.1</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#59023</td>
			<td>The ubiquitously expressed sqh promoter/enhancer drives expression of a fragment of Moesin (that includes the actin binding sequences) tagged with GFPS65T.</td>
		</tr>
		<tr>
			<td>hemocyte specific serpent-Gal4 driver 
			;srp-Gal4;</td>
			<td>Generated by Katja Bruckner</td>
			<td>Generated by Katja Bruckner</td>
			<td>Expression of Scer\GAL4 fused to a polyA tail is under the control of 2 genomic sequences from upstream of Drosophila serpent. Ref: Br&#252;ckner, K., Kockel, L., Duchek, P., Luque, C.M., R&#248;rth, P., Perrimon, N. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell. 7 (1), 73&#8211;84, doi: 10.1016/j.devcel.2004.06.007 (2004).</td>
		</tr>
		<tr>
			<td>UAS-nuclearRFP 
			w1118;;P{UAS-RedStinger}6</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#8545 or #8547</td>
			<td>UAS regulatory sequences drive expression of the DsRed.T4 form of RFP which is tagged at the C-terminal end with a nuclear localisation signal</td>
		</tr>
		<tr>
			<td>UAS-cytoplasmicGFP 
			;;P{UAS-GFP.S65T}</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>Multiple stocks available (e.g. #1522)</td>
			<td>Expression of the S65T version of GFP by UAS regulatory sequences; the S65T variant exhibits increased brightness.</td>
		</tr>
		<tr>
			<td>UAS-photoconvertibleKaede 
			w1118;; P{UAS-Kaede.A}3</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#26161</td>
			<td>Kaede protein emits bright green fluorescence after synthesis, but changes efficiently to a bright stable red fluorescence on irradiation with UV.</td>
		</tr>
		<tr>
			<td>GFP-tagged spaghetti squash 
			w1118;;P{sqh-GFP.RLC}</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#57145</td>
			<td>The sqh coding region, which is tagged at the C-terminal end with a T:Avic\GFPS65T tag, is expressed under the control of the natural sqh promoter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ingredients for fly food media</td>
			<td></td>
			<td></td>
			<td>Fly food media is made according to standard procedures (see Greenspan, R. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Press. 1-191 pp.)</td>
		</tr>
		<tr>
			<td>maize</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>soya flour</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>malt extract</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>molasses</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Difco agar</td>
			<td>BD Biosciences, Fisher Scientific</td>
			<td>DF0142-15-2</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma</td>
			<td>402907</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Nipagen</td>
			<td>Sigma</td>
			<td>79721</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Dried baker&#39;s yeast</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sample preparation and mounting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Fine sable paintbrush</td>
			<td>Daler-Rowney (or equivalent)</td>
			<td>#0 or 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific (or Fine Science Tools)</td>
			<td>NC9404145</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Glass bottomed dishes for imaging</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>We suggest using 35mm petri dishes, with at least a 10mm Microwell, 0.085-0.13mm cover glass, uncoated. Dishes with larger microwells will enable increasing numbers of pupae to be mounted and imaged in a single experiment.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma</td>
			<td>51730-5ML</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Double sided sticky tape (e.g. Scotch)</td>
			<td>Agar Scientific</td>
			<td>AGG263</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>50ml tube (for heptane glue)</td>
			<td>Falcon tubes from Fisher Scientific</td>
			<td>14-432-22</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Agar Scientific</td>
			<td>AGL4244</td>
			<td>For dissection of Drosophila pupae</td>
		</tr>
		<tr>
			<td>Dissecting stereo microscope with brightfield</td>
			<td>Leica (or equivalent)</td>
			<td>M50</td>
			<td>For dissection of Drosophila pupae</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>John Weiss International</td>
			<td>103123</td>
			<td>Miniature Research Scissors (straight)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Laser ablation and imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitogen ablation laser</td>
			<td>Spectra-Physics (or Andor equivalent)</td>
			<td>Model VSL-337ND-S</td>
			<td>For wounding, this should be attached to a widefield imaging system</td>
		</tr>
		<tr>
			<td>Multilaser confocal laser-scanning microscope (CLSM)</td>
			<td>Leica (or equivalent)</td>
			<td>TCS AOBS SP8 or SP5-II attached to a Leica DMi8 inverted epifluorescence microscope (or equivalent)</td>
			<td>Ideally including a motorised stage for multi-site and &#39;mosaic&#39; scanning, plus &#8216;hybrid&#8217; GaAsP detectors (that offer much greater sensitivity and boosting of low signal)</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Life Imaging Services (or equivalent)</td>
			<td>&#34;Microscope Temperature Control System&#34;</td>
			<td>Attached to Confocal microscope for temperature control during imaging</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FRAP software module</td>
			<td>Leica (or equivalent)</td>
			<td>CLSM FRAP software module</td>
			<td>For performing photoconversion of photoconvertible fluorophores such as Kaede</td>
		</tr>
		<tr>
			<td>ImageJ (image analysis software)</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Schneider, C.A., Rasband, W.S., Eliceiri, K.W. &#34;NIH Image to ImageJ: 25 years of image analysis&#34;. Nature Methods 9, 671-675, 2012.</td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;Manual Tracking&#34;</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.net/Manual_Tracking</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;TrackMate&#34;</td>
			<td>ImageJ, NIH</td>
			<td>https://imagej.net/TrackMate</td>
			<td>Tinevez, JY.; Perry, N. &#38; Schindelin, J. et al. (2016), &#34;TrackMate: An open and extensible platform for single-particle tracking.&#34;, Methods 115: 80-90, PMID 27713081</td>
		</tr>
		<tr>
			<td>Volocity (high performance 3D imaging software)</td>
			<td>Perkin Elmer</td>
			<td>Volocity 6.3</td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>IMARIS (image analysis software)</td>
			<td>Bitplane</td>
			<td>IMARIS for Cell Biologists</td>
			<td>For image analysis</td>
		</tr>
	</tbody>
</table>

long-term in vivo tracking of inflammatory cell dynamics within drosophila pupae

ऊतक क्षति के लिए तेजी से भड़काऊ प्रतिक्रिया के दौरान, जन्मजात प्रतिरक्षा प्रणाली की कोशिकाओं को जल्दी चोट साइट के लिए भर्ती कर रहे हैं । घाव पर एक बार, जंमजात प्रतिरक्षा कोशिकाओं ऐसे संक्रमण से लड़ने के रूप में आवश्यक कार्यों, की एक संख्या प्रदर्शन, गल मलबे समाशोधन, और मैट्रिक्स जमाव उत्तेजक । आदेश में पूरी तरह से विविध संकेतन घटनाओं है कि इस प्रतिरक्षा प्रतिक्रिया को विनियमित समझने के लिए, यह (और बातचीत है कि के बीच होने के जटिल व्यवहार का पालन करने के लिए महत्वपूर्ण है) vivo में एकाधिक सेल वंश, और वास्तविक समय में, उच्च के साथ spatio-लौकिक संकल्प । ऑप्टिकल translucency और Drosophila भ्रूण की आनुवंशिक पथ की एक अमूल्य मॉडल के रूप में Drosophila की स्थापना की है जीने की छवि और काटना के तंत्र सहित भड़काऊ कोशिका व्यवहार के बुनियादी पहलुओं, विकासात्मक फैलाव, अपोप्तोटिक लाशों और/या माइक्रोबियल रोगजनकों की मंजूरी, और घावों को भर्ती । हालांकि, अधिक हाल के काम अब दिखा दिया है कि Drosophila जीवन चक्र में एक बहुत बाद में मंच को रोजगार- Drosophila pupa-बेहतर RNAi दक्षता, अब इमेजिंग अवधि सहित अलग लाभ, की एक संख्या प्रदान करता है, और काफी अधिक प्रतिरक्षा सेल संख्या । यहाँ हम इमेजिंग घाव की मरम्मत और लाइव Drosophila प्यूपा में उच्च spatio-लौकिक संकल्प पर जुड़े भड़काऊ प्रतिक्रिया के लिए एक प्रोटोकॉल का वर्णन. दोनों पुनः epithelialization और सूजन की गतिशीलता का पालन करने के लिए, हम दोनों उपकला और जंमजात प्रतिरक्षा कोशिकाओं के लिए vivo फ्लोरोसेंट मार्करों में विशिष्ट की एक संख्या का उपयोग करें । हम भी विशिष्ट प्रतिरक्षा कक्ष सबसेट का पालन करने के लिए, उनके व्यवहार को ट्रैक करने के लिए, और से हल करने के लिए, चोट साइट, जैसे Kaede के रूप में फोटो-परिवर्तनीय fluorophores की प्रभावशीलता का प्रदर्शन ।

Watch this Scientific Journal Video about Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae at JoVE.com

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

यहाँ हम लाइव इमेजिंग घाव मरम्मत और vivo मेंउच्च spatio-लौकिक समाधान पर जुड़े भड़काऊ प्रतिक्रिया के लिए एक प्रोटोकॉल प्रस्तुत करते हैं । इस विधि Drosophila विकास के pupal चरण का उपयोग करने के लिए दीर्घकालिक इमेजिंग सक्षम और समय के साथ विशिष्ट कोशिका आबादी की ट्रैकिंग और कुशल RNAi-मध्यस्थता जीन निष्क्रियता के साथ संगत है ।

Vivo में Drosophila प्यूपा के भीतर भड़काऊ कोशिका गतिशीलता की ट्रैकिंग लंबी अवधि

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

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Immunology and Infection

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

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