The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

Invertebrate Tierstudien durchgeführt, hier sind in den Vereinigten Staaten nicht geregelt; daher die Institutional Animal Care und Use Committee hat keine Befugnis zur Überprüfung solcher Protokolle. Anstelle dieser nicht unter die Zuständigkeit der Regulierung in den Vereinigten Staaten fallen, so die Autoren hiermit, dass diese Studien wurden mit aufrichtigen Bemühungen im Hinblick auf die ethische Nutzung, Pflege und Behandlung dieser Tiere durchgeführt wird, wurde die Anzahl der Individuen minimiert und diese Bemühungen mit der Basler Deklaration und des Internationalen Rates für Versuchstierkunde (ICLAS) Ethik-Richtlinien stehen im Einklang. 1. Doryteuthis pealeii (D. pealeii) Dissection <ol><li> Kauf enthauptet Tintenfisch D. pealeii von der Marine Ressourcen Abteilung am Marine Biological Laboratory in Woods Hole, MA oder einer Frischfischmarkt. Verwenden Sie keine Proben, die um verrundet oder zuvor geändert wurden, um dieses Verfahren anzuwenden. Insbesondere ist die Chromatophore Schicht muss noch auf die Probe intakt sein. <ol><li> Wenn die Tiere nicht sofort verwendet werden, lagern Sie sie bei -20 ° C bis zur Verwendung. Um die Proben aufzutauen, legen Sie sie in Wasser bei Raumtemperatur für 1 Stunde vor der Präparation. </li></ol></li><li> Mit Edelstahl gerade fein-Punkt-Dissektion Schere entlang der hinteren Seite des Tieres geschnitten. Beginnen an der Basis des ventralen Mantel und an dem Rippenbereich endet. </li><li> Entfernen Sie alle inneren Organe (Magen, Kiemen, Tinte Drüse, Fortpflanzungsorgane, systemische Herz und brachial Herz) von ihnen heben mit einer Pinzette zu sezieren und Abtrennen ihre Bindegewebe Dissektion Schere. Entsorgen Organe in einem Probenbeutel. </li><li> Verwenden Sie sezieren T-Pins den Mantel an Pin (fin Seite nach oben) in einem Standard-11-1 / 2 &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;pan Aluminium Dissektion eine flexible Präparation Pad enthält. Versenken des Gewebes in 250 ml Meerwasser, das durch eine Einweg-Polystyrol-Vakuum-Filtrationssystem gefiltert wurdeenthaltend 0,2 &amp; mgr; m Porengrößen. </li><li> den epidermalen Hautschicht (Deckfarbe) mit Pinzette, die Chromatophoren haltige Hautschicht intakt bleibt vorsichtig entfernen. Sobald die epidermale Schicht entfernt wird, entsorgen Sie sie in einem Probenbeutel. </li><li> Präparieren klein (1 cm x 1 cm) Abschnitte der Chromatophoren-Schicht mit einer Pinzette und Schere Dissektion. Die Proben werden in 1,5 ml Mikrozentrifugenröhrchen (füllen die Hälfte jedes Leerrohr mit dem sezierten Gewebe). </li><li> Hinzufügen 500 ul gefiltertes Seewasser mit dem Gewebe enthaltenden Röhrchen. Die Proben können über Nacht bei 4 ° C gelagert werden. </li></ol> 2. Isolierkanne Chromatophor Pigmentgranula <ol><li> Tissue Verdauung <ol><li> Herstellung von Papain / Kollagenase <ol><li> Man wiegt 30 mg Kollagenase und 5 mg Papain mit einem 4 &quot;x 4&quot; niedrigen Stickstoff wiegen Papier. Bringen Sie diese in einem 50 ml-Polypropylen-Zentrifugenröhrchen mit Schraubverschluss. </li><li> Messen Sie 10 ml deionized Wasser mit einer Messpipette. Fügen Sie direkt Papain / Kollagenase Fläschchen enthält. Vortex auf die höchste Einstellung, bis die Lösung klar ist. </li></ol></li><li> Entfernen extrazelluläre Anhäufung <ol><li> Zentrifugieren des gesammelten chromatophore Gewebe aus Schritt 1.7 für 5 min bei 14.000 x g. Entsorgen Sie die obere Schicht aus Meerwasser in einen Kunststoffabfallbehälter. </li><li> In 500 ul der Papain / Kollagenase-Lösung auf das Gewebe ein P1000 variablen Volumen Einkanal-Mikropipette. Vortex jede Probe für 1-2 Minuten auf die höchste Einstellung. </li><li> Beschallen die Proben 5 min bei einer Frequenz von 40 kHz Zellen aus dem umgebenden Gewebe zu trennen. </li><li> Zentrifuge für 5 min bei 14.000 x g. Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. </li><li> Zentrifugieren des gesammelten chromatophore Gewebe für 5 min bei 14.000 x g. Entsorgen Sie die obere Schicht aus Meerwasser in einen Kunststoffabfallbehälter. Wiederholen Sie noch einmal. </li><li> Entfernen Sie manuell alle verbleibenden großen Gewebe secgen, die in diesem Prozess mit einer Pinzette verdauen nicht vor dem nächsten Schritt fortfahren. </li></ol></li></ol></li><li> Isolieren der Pigmentkörnchen <ol><li> Herstellung von Homogenisierungspuffer <ol><li> Wiegen 2,38 g (4- (2-Hydroxyethyl) -1-piperazinethansulfonsäure) (HEPES, 100 mM), 0.095 g Magnesiumchlorid (10 mM), 0,856 g Kalium-aspartat (50 mM) und 0,015 g Dithiothreitol (1 mM). In einen 150 ml Polystyrolbehälter mit Schraubverschluss. In einer kommerziellen Mini-Tablet-Protease-Inhibitor. </li><li> Messung der 100 ml entionisiertes Wasser mit einem Meßzylinder verwendet wird. Fügen Sie direkt in den Behälter, der die Homogenisierung Salze enthält. Vortex, bis die Lösung klar ist. </li></ol></li><li> Homogenisieren von Pigmentgranulaten <ol><li> Zentrifugieren jede Probe aus Schritt 2.1 bei 14000 × g für 5 min. Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. Diese Probe sollte frei von extrazellulären Gewebe sein. </li><li> In 500 &amp;# 181; l Homogenisierungspuffer in jedes Röhrchen und Vortex für 1-2 min je gründlich die Proben mischen. Beschallen die Proben für 30 Minuten (~ 40 kHz) und folgen mit 5 min Zentrifugation bei 14.000 x g. </li><li> Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter, und wiederholen Sie 2-3 mal den vorherigen Schritt die vollständige Verdauung der chromatophore sacculus und Protein Anbindehaltung zu gewährleisten. Die verbleibende Lösung sollte eine hellgelbe Überstand enthalten und eine rot gefärbte Pellet die isolierten Pigmentkörnchen enthalten. </li></ol></li></ol></li></ol> 3. Pigmentextraktion <ol><li> Herstellung von saurem Methanol (HCl-MeOH) <ol><li> Vorsichtig 25 ul konzentriert (36,5 bis 38% (w / w)), Salzsäure (HCl) zu 5 ml Methanol (MeOH). Lagern Sie das HCl-MeOH-Lösung in einem Schraubdeckel Glasprobenfläschchen. Vorsichtig schwenken, um die Lösung gleichmäßig mischen. HINWEIS: Je saurer die Lösung ist, desto mehr Pigment wird während der fi extrahiert werdenerste Extraktion. </li></ol></li><li> Extraktion von Pigment <ol><li> Zentrifugieren Sie die Pigmentgranulatsuspension (von 2.2.2) für 5 min bei 14.000 xg und entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. </li><li> In 500 ul der HCl-MeOH-Lösung und Vortex jede Probe für ~ 3-4 min. Beschallen die Proben für 10 min (~ 40 kHz), dann zentrifugieren für 5 min bei 14.000 x g. </li><li> Mit Hilfe einer Transferpipette, sammeln Sie den Überstand in einer 1-Dram-Schraubdeckel-Glasfläschchen. Der Überstand sollte in der Farbe dunkelrot sein. </li><li> Wiederholen Sie den Extraktionsschritt 3.2.2 bis keine weitere Farbe extrahiert (~ 4-5 Wäschen). HINWEIS: Jede Extraktion wird ~ 1,5 ml Pigment ergeben. Das verbleibende farblose Pellet wird das Pigment extrahierte Granulat. Hinweis: beide farblos Granulat und extrahiert Pigmente können in Wasser bei 4 ° C bis zur weiteren Verwendung gelagert werden. </li></ol></li></ol> 4. Charakterisierung gesamten Extraktionsverfahren <ol><li> Scannen Electron Mikroskopie <ol><li> Bild das Granulat (nicht umgesetztes aus Schritt 2.2.1 und pigment extrahiert aus Schritt 3.2.3) unter Verwendung von Rasterelektronenmikroskopie (SEM), um die Reinheit des Extraktionsprozesses zu überprüfen. </li><li> Um Granulatproben für die Bildgebung, erste Zentrifuge beide nicht umgesetzten Granulat und Pigment extrahiert Granulat für 5 min bei 14.000 · g, und entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter herzustellen. Re-suspendieren diese in 1 ml Ethanol und Ablagerung 2-3 Tropfen der Granulatsuspension auf Aluminium SEM Stubs eine Transferpipette. </li><li> über Nacht, nachdem die Proben trocknen, Sputter-Mantel mit Gold-Platin-Beschichtung für 3 min eine 15 nm Beschichtung zu erreichen. </li><li> Bild unter Verwendung eines SEM mit einer Schottky-Emitter und einem Sekundärelektronendetektor. Verwenden Sie einen 5-7 kV Strahlspannung und einen Arbeitsabstand zwischen 8 und 9 mm Bild diese Materialien. </li></ol></li><li> UV-VIS (UV-vis) spektrophotometrische Messungen <ol><li> Überwachen Sie die Absorption proDateien für alle drei Bedingungen (nicht umgesetztes Granulat aus 2.2.2, extrahiert Pigment aus 3.2.3 und Pigment extrahiert Granulat aus 3.2.4) mit einem Doppelstrahl-Spektrophotometer 200-800 nm verwendet wird. </li><li> Sammeln Sie eine Leermessung VE-Wasser in einer 1-ml-Küvette für den nicht umgesetzten Granulat und das Pigment extrahiert Granulat verwendet wird. Sammeln Sie UV-Vis-Absorptionsspektren für beide Proben. </li><li> Sammeln Sie eine leere Messung der sauren Methanollösung in einer 1-ml-Küvette für das extrahierte Pigment verwendet wird. Sammeln Sie UV-Vis-Absorptionsspektrum für die Probe. </li><li> Bestimmen maximale Wellenlänge jeder Probe durch die Wellenlänge identifiziert, an dem die Absorptionsspitze ihre maximale relative Höhe erreicht. </li></ol></li></ol>

<ol>
	<li>Bell, G. R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatophore+radial+muscle+fibers+anchor+in+flexible+squid+skin.">Chromatophore radial muscle fibers anchor in flexible squid skin.</a> Invertebr Biol. 132 (2), 120-132 (2013).</li><li>Crookes, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectins:+The+unusual+proteins+of+squid+reflective+tissues.">Reflectins: The unusual proteins of squid reflective tissues.</a> Science. 303 (5655), 235-238 (2004).</li><li>Deravi, L. F., 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+structure-function+relationships+of+a+natural+nanoscale+photonic+device+in+cuttlefish+chromatophores.">The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.</a> J. R. Soc. Interface. 11 (93), 1-9 (2014).</li><li>M&#228;thger, L. M., Denton, E. J., Marshall, N. J., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+behavioural+functions+of+structural+coloration+in+cephalopods.">Mechanisms and behavioural functions of structural coloration in cephalopods.</a> J. R. Soc. Interface. 6 (2), 149-163 (2009).</li><li>M&#228;thger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+basis+for+camouflaged+polarized+light+communication+in+squid.">Anatomical basis for camouflaged polarized light communication in squid.</a> Biol. Lett. 2 (4), 494-496 (2006).</li><li>M&#228;thger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malleable+skin+coloration+in+cephalopods:+selective+reflectance,+transmission+and+absorbance+of+light+by+chromatophores+and+iridophores.">Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores.</a> Cell Tissue Res. 329 (1), 179-186 (2007).</li><li>Cloney, R. A., Brocco, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatophore+Organs,+Reflector+Cells,+Iridocytes+and+Leucophores+in+Cephalopods.">Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods.</a> Amer. Zool. 23 (3), 581-592 (1983).</li><li>Hanlon, R. T., Messenger, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+coloration+in+young+cutttlefish+(Sepia+officinalis+L)-+The+morphology+and+development+of+body+patterns+and+their+relation+to+behaviour.">Adaptive coloration in young cutttlefish (Sepia officinalis L)- The morphology and development of body patterns and their relation to behaviour.</a> Philos Trans R Soc Lond B Biol Sci. 320 (1200), 437-487 (1988).</li><li>Sutherland, R. L., M&#228;thger, L. M., Hanlon, R. T., Urbas, A. M., Stone, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cephalopod+coloration+model.+II.+Multiple+layer+skin+effects.">Cephalopod coloration model. II. Multiple layer skin effects.</a> J. Opt. Soc. Am. 25 (8), 2044-2054 (2008).</li><li>Florey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+function+of+cephalopod+chromatophores.">Ultrastructure and function of cephalopod chromatophores.</a> Amer. Zool. 9 (2), 429-442 (1969).</li><li>Florey, E., Kriebel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+chromatophore+muscle+fibers+of+squid,+Loligo+opalescens,+to+nerve+stimulation+and+drugs.">Electrical and mechanical responses of chromatophore muscle fibers of squid, Loligo opalescens, to nerve stimulation and drugs.</a> Z. Vergl. Physiol. 65 (1), 98-130 (1969).</li><li>Williams, T. L., 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=Contributions+of+Phenoxazone-Based+Pigments+to+the+Structure+and+Function+of+Nanostructured+Granules+in+Squid+Chromatophores.">Contributions of Phenoxazone-Based Pigments to the Structure and Function of Nanostructured Granules in Squid Chromatophores.</a> Langmuir. , (2016).</li><li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ommochrome+pigmentation+of+the+linea+and+rosa+seasonal+forms+of+Precis+coenia+(Lepidoptera:+Nymphalidae).">Ommochrome pigmentation of the linea and rosa seasonal forms of Precis coenia (Lepidoptera: Nymphalidae).</a> Arch. Insect. Biochem. Physiol. 36 (3), 215-222 (1997).</li><li>Van Den Branden, C., Decleir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+Chromatophore+Pigments+in+the+Skin+of+the+Cephalopod+Sepia+Officinalis.">A Study of the Chromatophore Pigments in the Skin of the Cephalopod Sepia Officinalis.</a> L. Biol. Jb. Dodonaea. 44 (2), 345-352 (1976).</li><li>Aubourg, S., Torres-Arreola, W., Trigo, M., Ezquerra-Brauer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Jumbo+Squid+Skin+Pigment+Extract+and+its+Antioxidant+Potential+ina+Marine+Oil+System.">Characterization of Jumbo Squid Skin Pigment Extract and its Antioxidant Potential ina Marine Oil System.</a> Eur. J. Lipid Sci. Technol. 118, (2016).</li></ol>

We have nothing to disclose.

Wir haben ein Verfahren zum Extrahieren von Pigmenten squid chromatophore Granulat demonstriert. Durch speziell auf die Granulat-Targeting, unser Ziel ist es, ihre Rolle bei der Vermittlung der adaptive Färbung zu bestimmen. Diese Methode unterscheidet sich von früheren Berichten entworfen Kopffüßer Pigmente mit Bulk - Gewebeproben 14 oder gefriergetrocknet Haut 15 zu charakterisieren. Während dieses Protokolls auf Extrahieren chromatophore Pigmente wirksam ist, wi...

Kopffüßer wie Tintenfische, Tintenfische und Kraken haben die Fähigkeit, dynamisch ihr Aussehen verändern , für Tarnen und Signalisierung. 1-6 Diese Fähigkeit durch die selektive Flächenausdehnung von pigmentierten Organe teilweise als Chromatophoren bekannt unterstützt wird. 4,7-9 Chromatophoren Weich Aktoren , die ein Netzwerk von nanostrukturiertem Pigmentgranulaten in einem cytoelastic sacculus beschränkt enthalten , die radial von Muskelfasern verankert ist. 1,3 Da sie betätigt werden, Chromatophoren um 500% in präsentierten Oberfläche erweitern , um die Granulate im gesamten Organ verteilen. 3,7 10,11, Wenn diese Aktion über eine Anzahl von aufeinander abgestimmten Chromatophoren wird, wird die Gesamtfärbung des Tieres verändert. Während es bekannt ist, daß die Pigmentgranulate dieser Farbänderung beitragen, bleibt deren Zusammensetzung unbekannt. Wir beschreiben ein Verfahren zur Isolierung und Reinigung von chromatophore Pigmente, die für zukünftige Studien Zusammensetzungs angepasst werden kann. 3,12 Chromatophor haltiges Gewebe durch sorgfältige Exstirpation von der Kopffüßer geerntet wird. Eine Verdauung und Homogenisierungsverfahren wird dann verwendet, um das umgebende Gewebe zu dissoziieren und die chromatophore Zellen trennen. Die nanostrukturierten Granula werden dann isoliert und gereinigt aus den verbleibenden Chromatophoren wiederholt unter Verwendung von Beschallung und Zentrifugation. Nach der Reinigung werden Pigmente aus dem Granulat in einem Verfahren extrahiert , das aus der Gewinnung von sichtbaren Farbe aus Schmetterlingsflügeln mit saurem Methanol - Lösungen. 13 Rasterelektronenmikroskopie (SEM) und Spektrometrie werden verwendet , um zu bestätigen , dass die Chromatophoren - Pigmente erfolgreich extrahiert angepasst ist , dieser Prozess. Diese Methode beschreibt die Isolierung von chromatophore Granulat, das verwendet wird, um die molekularen Beiträge zu c zu erkundenoloration in Kopffüßer. 12 kleine Molekül - Extraktionen von ganzen Tieren kann oft ein langer und mühsamer Prozess sein. Das Ziel hier ist es, zukünftige Forscher einer effektiven und einfachen Protokoll für den Erwerb von Pigmenten aus den nanostrukturierten Granulat in Kopffüßer zu informieren.

<table border="0" cellpadding="0" cellspacing="0" width="611">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">9001-12-1</td>
			<td style="width:205px;">No hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3482-12-3</td>
			<td>Irritant, acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">7365-45-9</td>
			<td>Mild irritant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrochloric acid</td>
			<td style="width:71px;">EMD Chemicals</td>
			<td style="width:119px;">7647-01-0</td>
			<td>Corrosive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">k-Aspartate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1115-63-5</td>
			<td>Reacts violently with oxidants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnesium Chloride</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">7786-30-3</td>
			<td>Mild eye irritant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:71px;">Pharmco-AAPER</td>
			<td style="width:119px;">67-56-1</td>
			<td>Highly flammable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mini tablet prtoease inhibitor</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">469315-90-01</td>
			<td>Corrosive to metal and skin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Papain</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">9001-73-4</td>
			<td>Irritant&#160;</td>
		</tr>
	</tbody>
</table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Chromatophoren werden aus dem D. seziert pealeii dorsal Mantel (1A, 1B). Sobald sie entfernt werden, werden Chromatophoren lysiert und gereinigt Zyklen Zentrifugation und Waschen der pigmentierten Granulate (2A, 2B) zu isolieren geführt. Sauren Methanollösungen (HCl-MeOH) werden verwendet , um das Pigment aus dem Granulat (2C) zu extrahieren, um ein lösliches Pigment Extrakt und unl?...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Ein Protokoll für die Extraktion von Pigmenten aus den nanostrukturierten Granulat in Tintenfisch Doryteuthis pealeii Chromatophoren vorgestellt.

Ein Verfahren zum Extrahieren Pigmente aus Squid<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

a-method-for-extracting-pigments-from-squid-doryteuthis-pealeii

Research

JoVE Journal

Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K Aufrufe.  University of New Hampshire.  Ein Protokoll für die Extraktion von Pigmenten aus den nanostrukturierten Granulat in Tintenfisch Doryteuthis pealeii Chromatophoren vorgestellt. 

Serie: A Method for Extracting Pigments from Squid Doryteuthis pealeii

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

Extrahieren von DNA aus dem Gut Mikroben der Termite (Zootermopsis Angusticollis) und visualisieren Gut Mikroben

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

Forschung

Biologie

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Eine einfache Methode für genaue Genotypisierung

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Method for Culture of Early Hühnerembryonen ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Verwendung von Drehstabtest als Methode für die qualitative Analyse von Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

A Lectin HPLC-Methode zur selektiven glykosylierte Peptide aus komplexen biologischen Proben Enrich

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

Beziehen Blutzellen aus dem hawaiianischen Bobtail Squid Euprymna scolopes Und die Beobachtung ihrer Einhaltung Symbiotic und Non-symbiotischen Bakterien

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

A Simple Protocol für das Extrahieren Hämozyten von Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Eine praktische und neuartige Methode, um genomische DNA aus Blutentnahme Kits Auszug für Plasma Protein Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Kultur Verfahren zur embryonalen Maus Herz

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...