Name: non-terminal blood sampling techniques in guinea pigs
Uploaded: 2014-10-11T14:00:00
Description: Watch this Scientific Journal Video about Non-Terminal Blood Sampling Techniques in Guinea Pigs at JoVE.com

Vi anerkender den dygtige bistand fra Annie B. Kristensen, Elisabeth V. Andersen, Belinda B. Bringtoft og animalske pedeller på Institut for Eksperimentel Medicin, Frederiksberg Campus, Det Sundhedsvidenskabelige Fakultet, Københavns Universitet, Danmark. Jacob Lønholdt er takkede for hans fremragende teknisk bistand filme procedurerne. Denne undersøgelse blev støttet delvist af LIFEPHARM Center for in vivo Farmakologi og Københavns Universitet.

De viste procedurer blev udført i henhold til protokoller, der er godkendt af den danske Dyreforsøg inspektorat under Ministeriet for Fødevarer, Landbrug og Fiskeri. 1. Blodprøvetagning fra Lateral saphenus eller Tarsal Vein <ol><li> Udfør denne procedure uden bedøvelse og med hjælp fra en efterforsker, en håndtering af marsvin, og en opsamling af prøven. </li><li> Barber tarsal region bagbenet med en elektrisk barbermaskine, indtil vener er synlige. Sørg for, at området er stor...

<ol>
	<li>DeOgburn, 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=Effects+of+increased+dietary+cholesterol+with+carbohydrate+restriction+on+hepatic+lipid+metabolism+in+Guinea+pigs.">Effects of increased dietary cholesterol with carbohydrate restriction on hepatic lipid metabolism in Guinea pigs.</a> Comp.Med. 62 (2), 109-115 (2012).</li><li>Fernandez, M., Volek, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guinea+pigs+a+suitable+animal+model+to+study+lipoprotein+metabolism,+atherosclerosis+and+inflammation.">Guinea pigs a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation.</a> Nutr Metab (Lond). 3 (17), (2006).</li><li>Frikke-Schmidt, H., Tveden-Nyborg, P., Birck, M. M., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dietary+fat+and+cholesterol+exacerbates+chronic+vitamin+C+deficiency+in+guinea+pigs.">High dietary fat and cholesterol exacerbates chronic vitamin C deficiency in guinea pigs.</a> Br J Nutr. 105 (1), 54-61 (2011).</li><li>Hem, A., Smith, A. J., Solberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saphenous+vein+puncture+for+blood+sampling+of+the+mouse,+rat,+hamster,+gerbil,+guinea+pig,+ferret+and+mink.">Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink.</a> Lab Anim. 32 (4), 364-368 (1998).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+guidelines+and+recommendations.">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li><li>Ye, P., Cheah, I. K., Halliwell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diets+and+pathology+in+the+guinea+pig+Atherosclerosis+or+liver+damage.">High fat diets and pathology in the guinea pig Atherosclerosis or liver damage.</a> Biochim Biophys Acta. 1832 (2), 355-364 (2013).</li><li>Tveden-Nyborg, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+vitamin+C+deficiency+during+pregnancy+persistently+impairs+hippocampal+neurogenesis+in+offspring+of+guinea+pigs.">Maternal vitamin C deficiency during pregnancy persistently impairs hippocampal neurogenesis in offspring of guinea pigs.</a> PLoS One. 7 (10), 12-18391 (2012).</li><li>Schjoldager, J. G., Tveden-Nyborg, P., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+maternal+vitamin+C+deficiency+overrides+preferential+fetal+ascorbate+transport+but+does+not+influence+perinatal+survival+in+guinea+pigs.">Prolonged maternal vitamin C deficiency overrides preferential fetal ascorbate transport but does not influence perinatal survival in guinea pigs.</a> Br J Nutr. 110 (9), 1573-1579 (2013).</li><li>Greulich, 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=Secretory+products+of+guinea+pig+epicardial+fat+induce+insulin+resistance+and+impair+primary+adult+rat+cardiomyocyte+function.">Secretory products of guinea pig epicardial fat induce insulin resistance and impair primary adult rat cardiomyocyte function.</a> J Cell Mol Med. 15 (11), 2399-2410 (2011).</li><li>Swifka, J., Weiss, J., Addicks, K., Eckel, J., Rosen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+fat+from+guinea+pig+a+model+to+study+the+paracrine+network+of+interactions+between+epicardial+fat+and+myocardium.">Epicardial fat from guinea pig a model to study the paracrine network of interactions between epicardial fat and myocardium.</a> Cardiovasc Drugs Ther. 22 (2), 107-114 (2008).</li><li>Pilny, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hematology+of+rodent+species.">Clinical hematology of rodent species.</a> Vet.Clin North Am Exot Anim Pract. 11 (3), 523-vii (2008).</li><li>Franco, N. H., Olsson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientists+and+the+3Rs:+attitudes+to+animal+use+in+biomedical+research+and+the+effect+of+mandatory+training+in+laboratory+animal+science.">Scientists and the 3Rs: attitudes to animal use in biomedical research and the effect of mandatory training in laboratory animal science.</a> Lab Anim. 48 (1), 50-60 (2014).</li><li>Van, L. 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=eAssessing+the+application+of+the+3Rs+a+survey+among+animal+welfare+officers+in+The+Netherlands.">eAssessing the application of the 3Rs a survey among animal welfare officers in The Netherlands.</a> Lab Anim. 47 (3), 210-219 (2013).</li><li>Sloan, R. 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=High+doses+of+ketamine-xylazine+anesthesia+reduce+cardiac+ischemia-reperfusion+injury+in+guinea+pigs.">High doses of ketamine-xylazine anesthesia reduce cardiac ischemia-reperfusion injury in guinea pigs.</a> J Am Assoc Lab Anim Sci. 50 (3), 349-354 (2011).</li><li>Jacobson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+anaesthetic+regimen+for+surgical+procedures+in+guinea+pigs.">A novel anaesthetic regimen for surgical procedures in guinea pigs.</a> Lab Anim. 35 (3), 271-276 (2001).</li><li>Saha, J. K., Xia, J., Grondin, J. M., Engle, S. K., Jakubowski, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hyperglycemia+induced+by+ketamine+xylazine+anesthesia+in+rats+mechanisms+and+implications+for+preclinical+models.">Acute hyperglycemia induced by ketamine xylazine anesthesia in rats mechanisms and implications for preclinical models.</a> Exp Biol Med(Maywood). 230 (10), 777-784 (2005).</li></ol>

Sammenlignet med mus og rotter, marsvin er bange af natur og skal behandles roligt og håndteres korrekt med henblik på at reducere stress. Ud fra et dyrevelfærdsmæssigt synspunkt, der handler med omhu, er naturligvis en prioritet; Dette vil imidlertid også reducere risikoen for angst-effekter på indsamlede data f.eks., øgede blodniveauer af kortikosteroider og glucose 12. Ligeledes vil daglige håndtering vænne dyrene til normale procedurer og kan tjene til at reducere stress yderligere. Prak...

Marsvin er en værdifuld og valideret eksperimentel dyremodel på grund af en række biologiske ligheder til mennesker, såsom et krav om en kosten forsyning af C-vitamin, sammenlignelig plasma lipoprotein enzymer og lipoprotein profiler, samt delte ligheder med menneskelig placentation og prænatal udvikling 1-3,7,8. Dette gør marsvin et attraktivt og velegnet model til at studere virkningerne af kost-associerede sygdomme såsom alkoholfri fedtlever sygdom, hjerte-kar-sygdomme og formodede virkninger af C-vitaminmangel, men også effekten af f.eks., Moderens kostintervention på udviklingslandene afkom. Gentagen blodprøvetagning er ofte nødvendig i både lang og kort sigt, in vivo-undersøgelser. På grund af artsspecifikke anatomiske forskelle er nødvendigt modulation af stikprøveudtagningsteknik for at opnå blodprøver i marsvin sammenlignet med andre gnavermodeller <sup&gt; 4,5. Dette omfatter variation i løbet af vener og arterier, og endvidere, at marsvin ikke har en hale, har således forskellige krav og muligheder for både prøveudtagning og håndtering. Prøveudtagning fra saphenous eller tarsal vene, inducerer minimal ubehag og stress i dyret og ikke kræver anæstesi 3,9. Denne teknik gør det muligt gentagne prøver af små mængder af blod (100-400 pi), f.eks. At bestemme biokemiske markører i plasma. Hvis en endnu mindre stikprøve er tilstrækkelig (50-100 ul), kan dette ganske let opnås ved at punktere ørevene. Denne teknik kan også udføres i bevidste dyr og er nyttige for eksempel ved måling af blodglukose 10,11. Prøveudtagning fra halsvenen muliggør indsamling af større mængder af blod (1-2 ml), men kræver anæstesi. Her præsenterer vi fire forskellige blodprøvetagning teknikker til enten bevidste eller bedøvede marsvin. Procedures er alle ikke-terminale procedurer, forudsat at prøvevolumener og antal prøver ikke overstiger retningslinjer for indsamling af blod i forsøgsdyr 6. Det er af hensyn til god videnskab samt dyrevelfærd, at stress bør holdes på et minimum. Følgelig kan kun veluddannede og kompetent personlig skal udføre procedurerne dyr.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:295px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:315px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electric shaver</td>
			<td style="width:295px;">Wella (Contura), Denmark</td>
			<td style="width:119px;">273207</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringes&#160;</td>
			<td style="width:295px;">B.Braun-Omnifix-F, Germany</td>
			<td style="width:119px;">9161406V</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Needles</td>
			<td style="width:295px;">BD Microlance,Spain</td>
			<td style="width:119px;">301156</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microvette collection tube&#160;</td>
			<td style="width:295px;">Sarstedt, D-51588N&#252;mbrecht, Germany</td>
			<td style="width:119px;">201,288,100</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Accu-Check analyzer</td>
			<td style="width:295px;">Roche A/S Diagnostics, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Heparin 5000 IE/ml</td>
			<td style="width:295px;">LEO Pharma, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Zoletil 50, Chemvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Narcoxyl vet 20 mg/ml, Intervet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Torbugesic vet 10 mg/ml, Scanvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Neutral eye ointment</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze or a cotton swab&#160;</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfectant (70% alcohol)</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
	</tbody>
</table>

non-terminal blood sampling techniques in guinea pigs

Marsvin har flere biologiske ligheder til mennesker og valideres eksperimentelle dyremodeller 1-3. Men brugen af ​​marsvin i øjeblikket udgør et relativt smalt område af forskning og beskrivende data om specifikke metode er tilsvarende knappe. De anatomiske træk af marsvin er lidt forskellig fra andre gnavermodeller dermed modulering af stikprøver at rumme artsspecifikke forskelle, f.eks. Sammenlignet med mus og rotter, er nødvendige for at opnå tilstrækkelige og høj kvalitet prøver. Da både lang og kort sigt in vivo-undersøgelser ofte kræver gentagne blodprøvetagning valg af teknik bør være godt overvejes for at reducere stress og ubehag i dyrene, men også for at sikre overlevelse, samt overholdelse af kravene til stikprøvestørrelse og tilgængelighed. Kan fås venøse blodprøver på en række steder i marsvin f.eks., Saphenous og jugularisvener, hver teknik indeholder både fordele og ulemper 4,5. Her præsenterer vi fire forskellige blodprøvetagning teknikker til enten bevidste eller bedøvede marsvin. Procedurerne er alle ikke-terminale procedurer, forudsat at prøvevolumener og antal prøver ikke overstiger retningslinjer for indsamling af blod i forsøgsdyr 6. Alle de beskrevne metoder er blevet grundigt afprøvet og anvendt til gentagen in vivo blodprøveudtagning i studier inden for vores forskning facilitet.

Fire forskellige tilgange til ikke-terminal in vivo blodprøvetagning teknikker i enten bevidste eller bedøvede marsvin er blevet præsenteret. Figur 1 viser forløbet af den laterale vena og tarsal vene i marsvin og eventuelle punktur. Ørevener synlige på den dorsale overflade af et marsvin øre er illustreret i figur 2. Disse vener kan anvendes til blodprøvetagning, hvor kun en meget lille mængde er påkrævet. Placeringen af forsøgskanin for jugularis blodprøvetagning...

Watch this Scientific Journal Video about Non-Terminal Blood Sampling Techniques in Guinea Pigs at JoVE.com

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Selvom en kendt model, marsvin i øjeblikket udgør en niche i forsøgsdyr videnskaber og begrænsede data om gennemførelsen af ​​de fleste procedurer. Her præsenterer vi fire forskellige tilgange til ikke-terminal in vivo blodprøvetagning teknikker i enten bevidste eller bedøvede marsvin.

Ikke-Terminal blodprøvetagning teknikker i marsvin

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

non-terminal-blood-sampling-techniques-in-guinea-pigs

Research

JoVE Journal

Biology

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Teknikker til analyse af ekstracellulær Vesikler anvendelse af flowcytometri

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Tørrede blod pletter - forberedelse og forarbejdning til brug i immunassays og molekylærbiologiske teknikker

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blodprøvetagning fra den laterale halevene af Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Micron-skala fænotyper teknikker af majs kardiovaskulære bundter baseret på X-ray Microcomputed tomografi

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Gonadektomi og blodprøvetagning procedurer i den lille størrelse Teleost Model japanske Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.