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

We acknowledge the skilful assistance of Annie B. Kristensen, Elisabeth V. Andersen, Belinda B. Bringtoft and the animal caretakers at the Department of Experimental Medicine, Frederiksberg Campus, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Jacob L&#248;nholdt is thanked for his excellent technical assistance in filming the procedures. This study was supported in part by the LIFEPHARM Centre for in vivo Pharmacology and University of Copenhagen.

The shown procedures were performed according to protocols approved by the Danish Animal Experimentation Inspectorate under the Ministry of Food, Agriculture and Fisheries.
1. Blood Sampling from the Lateral Saphenous or Tarsal Vein
<ol>
	<li>Perform this procedure without anaesthesia and the help of one investigator, one handling the guinea pig, and one collecting the sample.</li>
	<li>Shave the tarsal region of the hind leg with an electric shaver until the veins are visible. Ensure the ar...

<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>

Compared to mice and rats, guinea pigs are fearful by nature and must be approached calmly and handled correctly in order to reduce stress. From an animal welfare point of view, acting with care is naturally a priority; however, this will also reduce the risk of anxiety-associated effects on the collected data e.g., increased blood levels of corticosteroids and glucose12. Likewise, daily handling will habituate the animals to normal procedures and may serve to reduce stress even further. Practical exp...

The guinea pig is a valuable and validated experimental animal model due to a number of biological similarities to humans such as a requirement for a dietary supply of vitamin C, comparable plasma lipoprotein metabolizing enzymes and lipoprotein profiles, as well as shared similarities with human placentation and prenatal development1-3,7,8. This makes the guinea pig an attractive and suitable model for studying effects of diet-associated diseases such as non-alcoholic fatty liver disease, cardiovascular diseases and putative effects of vitamin C deficiency but also the effect of e.g., maternal dietary intervention on the developing offspring.
Repeated blood sampling is often required in both long and short term in vivo studies. Due to species-specific anatomical differences modulation of the sampling technique is necessary in order to obtain blood samples in guinea pigs compared to other rodent models4,5. This includes variation in the course of veins and arteries, and also that guinea pigs do not have a tail, thus have different requirements and options for both sampling and handling. Sampling from the saphenous or tarsal vein, induces minimal discomfort and stress in the animal and does not require anaesthesia3,9. This technique allows repeated samples of small amounts of blood (100-400 &#181;l), e.g., to determine biochemical markers in plasma. If an even smaller sample size is sufficient (50-100 &#181;l), this can quite easily be obtained by puncturing the ear vein. This technique can also be carried out in conscious animals and is useful for example when measuring blood glucose10,11. Sampling from the jugular vein allows for collection of larger amounts of blood (1-2 ml) but requires anaesthesia.
Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. It is in the interests of good science as well as of animal welfare that stress should be kept to a minimum. Consequently, only well-trained and competent personal should perform the animal procedures.

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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

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs have been presented. Figure 1 illustrates the course of the lateral saphenous and tarsal vein in the guinea pig and possible puncture sites. Ear veins visible on the dorsal surface of a guinea pig ear are illustrated in Figure 2. These veins can be used for blood sampling when only a very small volume is required. The position of the guinea pig for jugular...

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

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

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

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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.

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.

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.

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.

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.

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.