We thank Virginia Doherty and Junmei Yao for technical assistance. This research was funded by NIMH (R01 MH084966), and the U.S. Army Research Office and the Defense Advanced Research Projects Agency (grant W911NF-10-1-0059) to KAG.

All experiments were done using adult male Long-Evans rats. All procedures were in accordance with the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Massachusetts Institute of Technology and the Animal Care and Use Review Office of the USAMRMC.
1. Preparation
<ol>
	<li>Heparinise the catheter and syringe by placing the shielded needle in a 500 &#181;l tube containing heparin (1,000 USP units/ml) and then aspirating and expelling heparin solution through the needle.
	<ol>
		<li>Attach a butterfly catheter to the syringe. Keep the shield over the needle of the catheter to protect the sharp tip from damage.</li>
		<li>Withdraw a volume of heparin that is slightly greater than the volume of blood that will be collected. Detach the syringe and fill it with air.</li>
		<li>Re-attach the syringe to the catheter and use the air to expel excess heparin solution; ensure only trace amounts remain in the tubing, needle, and syringe.</li>
		<li>Place the sterile catheter, with the syringe still attached, on a sterile surface.</li>
	</ol>
	</li>
	<li>Quickly secure the rat in a clean cloth ensuring that fore- and hindpaws are in a comfortable position and breathing is unrestricted.
	<ol>
		<li>Secure the wrap with hook and loop tape; ensure that external genitalia are not constricted.</li>
		<li>Have an assistant gently and firmly restrain the rat (abdomen and base of tail) on a solid work surface with the tail hanging off the edge of the counter.</li>
	</ol>
	</li>
</ol>
2. Blood Sampling
<ol>
	<li>Immerse the tail in 42 &#176;C water for 40-50 sec to dilate blood vessels and dry the tail with a paper towel. Locate the tail vein to be bled (rotate the whole body with the tail to prevent twisting the tail). 
	Note: Sufficient warming of the tail is critical for the rapid collection of a blood sample. If the vasculature is constricted, proper placement of the catheter is difficult, and blood flow is vastly reduced. A heating pad may be used as an alternative to water immersion.</li>
	<li>Identify the sampling point. 
	&#8203;Note: The artery lies along the mid-dorsal aspect of the tail; do not use this for sampling.
	<ol>
		<li>Target either the left and right tail veins that lie lateral to the artery. Pigmentation of the tail, which varies by strain and increases with age, may obscure some of the vasculature. Target a portion of the vein in the lower portion of the tail.</li>
		<li>Wipe the target area with 2% chlorhexidine antiseptic solution.</li>
	</ol>
	</li>
	<li>Create negative pressure in the syringe and catheter by withdrawing the plunger from zero to approximately 50 &#181;l.</li>
	<li>Hold the tail gently and firmly near the tip to keep the tail straight throughout sample collection. Ensure that blood flow is not occluded by an overly tight grip.</li>
	<li>Slowly insert the catheter into the vein at a shallow angle approximately 5 cm from the tip of the tail. When the vein is penetrated, blood will flow into the catheter. Slowly withdraw the plunger of the syringe to collect the desired volume at a steady rate (~20 &#181;l per sec).
	<ol>
		<li>Consult the veterinary staff for information about the maximum blood volume that can be collected. The maximum amount of blood that should be collected depends on the weight and health status of the rat. Do not withdraw more than 15% of total blood volume in a 14 day period. 
		Note: Blood is much more difficult to collect from animals that were acutely stressed in the minutes prior to sample collection because stress hormones constrict the vasculature. For example, moving the rat&#39;s home cage to a novel room, taking several minutes to wrap the animal, or repeated insertion of the catheter into a vein are all likely to trigger an acute stress response.</li>
		<li>Facilitate blood flow by &#8216;milking&#39; the vein. Run a finger along the length of the vein, from the base towards the tip of the tail, but remain more than 2 cm from the tip of the inserted needle or the catheter may become dislodged from the vein.</li>
		<li>If blood cannot be successfully collected from the initial site of catheter penetration, re-insert the needle further up the vein. If blood was collected at the initial site, re-pressurize the needle by disconnecting and then reconnecting the catheter and syringe prior to re-insertion in the vein. In general, avoid additional penetrations.</li>
		<li>As multiple penetrations can cause tail vein collapse, in which the blood supply to the tail is cut off and the soft tail tissue becomes necrotized, euthanize the rat if there is tail vein collapse.</li>
	</ol>
	</li>
	<li>When adequate sample volume is collected, release pressure in the syringe by disconnecting and reconnecting the catheter. Aspirate slightly using the syringe plunger (~50 &#181;l), and withdraw the needle from the vein. 
	Note: If the needle is withdrawn without first releasing the pressure in the syringe, blood will drip from the needle.</li>
	<li>Briefly apply pressure to the insertion site to stop bleeding, and wipe the area with antiseptic solution. Return the rat to its home cage.</li>
</ol>
3. Processing the Blood Sample
<ol>
	<li>Aspirate air to ensure no blood remains inside the catheter needle, and use scissors to cut the catheter tubing just above the needle. Expel the blood into a sterile 1.5 ml microcentrifuge tube. 
	Note: If blood is pushed through the needle, the shearing force may cause red blood cells to rupture which can interfere with many downstream assays. Remove the needle to avoid hemolysis.
	<ol>
		<li>To collect blood plasma, use tubes that contain EDTA as an anticoagulant (here, use 10 &#181;l of 0.1 M EDTA for 200-400 &#181;l of blood; ensure the concentration of EDTA used does not interfere with the downstream assay) and place on ice.
		<ol>
			<li>Spin whole blood samples at 2,100 x g in a refrigerated centrifuge (4 &#176;C) for 10 min within 10 min of collection. Elute the plasma, avoiding disturbing the red and white blood cell layers.</li>
		</ol>
		</li>
		<li>To collect blood serum, place samples (without anticoagulant) at room temperature for up to 30 min to enable clotting. Spin the collection tubes in a refrigerated centrifuge (4 &#176;C) at 2,000 x g. The serum may then be eluted.</li>
	</ol>
	</li>
	<li>Use samples immediately, or store at -80 &#176;C for up to one year.</li>
</ol>

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	<li>Vausort, M., Wagner, D. R., Devaux, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Noncoding+RNAs+in+Patients+with+Acute+Myocardial+Infarction.">Long Noncoding RNAs in Patients with Acute Myocardial Infarction.</a> Circ Res. 115 (7), 668-677 (2014).</li><li>Shah, 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=Biomarkers+for+Early+Detection+of+Colorectal+Cancer+and+Polyps:+Systematic+Review.">Biomarkers for Early Detection of Colorectal Cancer and Polyps: Systematic Review.</a> Cancer Epidemiol Biomarkers Prev. 23 (9), 1712-1728 (2014).</li><li>Chan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+blood-based+protein+biomarker+strategies+in+the+study+of+psychiatric+disorders.">Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.</a> Prog Neurobiol. , (2014).</li><li>Cao, L., Li, L., Lin, D., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+learning+impairment+that+is+mediated+by+interleukin+1beta+in+rodents.">Isoflurane induces learning impairment that is mediated by interleukin 1beta in rodents.</a> PLoS One. 7 (12), e51431 (2012).</li><li>Culley, D. J., Baxter, M. G., Yukhananov, R., Crosby, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impairment+of+acquisition+of+a+spatial+memory+task+following+isoflurane-nitrous+oxide+anesthesia+in+rats.">Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats.</a> Anesthesiology. 100 (2), 309-314 (2004).</li><li>Vahl, T. 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=Comparative+analysis+of+ACTH+and+corticosterone+sampling+methods+in+rats.">Comparative analysis of ACTH and corticosterone sampling methods in rats.</a> Am J Physiol Endocrinol Metab. 289 (5), E823-E828 (2005).</li><li>Kalinichenko, L. S., Koplik, E. V., Pertsov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+rats+with+various+behavioral+characteristics+during+acute+emotional+stress.">Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.</a> Bull Exp Biol Med. 156 (4), 441-444 (2014).</li><li>McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+stress+hormones+in+health+and+disease:+Understanding+the+protective+and+damaging+effects+of+stress+and+stress+mediators.">Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators.</a> Eur J Pharmacol. 583 (2-3), 174-185 (2008).</li><li>Sanchez, A., Toledo-Pinto, E. A., Menezes, M. L., Pereira, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+norepinephrine+and+epinephrine+concentrations+in+adrenal+gland+of+the+rats+submitted+to+acute+immobilization+stress.">Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted to acute immobilization stress.</a> Pharmacol Res. 48 (6), 607-613 (2003).</li><li>Meyer, R. M., Burgos-Robles, A., Liu, E., Correia, S. S., Goosens, K. 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+ghrelin-growth+hormone+axis+drives+stress-induced+vulnerability+to+enhanced+fear.">A ghrelin-growth hormone axis drives stress-induced vulnerability to enhanced fear.</a> Mol Psychiatry. , (2013).</li><li>Knoll, A. T., Carlezon, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynorphin,+stress,+and+depression.">Dynorphin, stress, and depression.</a> Brain Res. 1314 (56-73), (2010).</li><li>Harvey, B. H., Brand, L., Jeeva, Z., Stein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical/hippocampal+monoamines,+HPA-axis+changes+and+aversive+behavior+following+stress+and+restress+in+an+animal+model+of+post-traumatic+stress+disorder.">Cortical/hippocampal monoamines, HPA-axis changes and aversive behavior following stress and restress in an animal model of post-traumatic stress disorder.</a> Physiol Behav. 87 (5), 881-890 (2006).</li></ol>

The authors have nothing to disclose.

Here, we describe a quick and simple procedure for obtaining a blood sample from a rat which offers significant advantages over other commonly used techniques. First, it does not require anesthesia, in contrast with sampling from the jugular vein or retroorbital sinus. When blood samples are collected surrounding behavioral procedures, administration of anesthetics is undesirable because it can interfere with learning and memory4,5. Second, it offers the ability to collect larger blood volumes than other venipuncture techniques, such as collection from the saphenous or dorsal pedal veins. Using the technique described here, up to 1.5 ml of blood may be collected from a rat at a single time point, a volume which readily allows multiple assays to be run in parallel. Finally, this procedure minimizes the potential for tissue damage compared to tail tip amputation or retroorbital bleeding. The use of this procedure facilitates compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, which require minimizing the pain and distress that result from laboratory procedures performed on animals.
It is recommended that investigators new to this method practice the restraint and tail bleeding techniques in order to minimize the time that experimental animals are restrained. Blood collected in less than 3 min from the initiation of restraint provides optimal results.
The protocol described here may be used for sampling 1 to 4 times per week, but no more than twice per day. While repeated blood collections may be performed, different sampling sites moving upwards from the base of the tail should be used, and the left and right tail veins should be alternated as sampling sites. The total blood volume of rodents is 6-7% of their body weight, and no more than 15% of the total blood volume should be collected within a 2 week period. Serum or plasma comprises approximately 40-60% of the collected sample volume.
Blood sampling via the lateral tail veins may also be performed in the mouse as described here with a few minor modifications. First, only small gauge (27 G) catheters may be used. Second, it is recommended to use a tube restrainer, rather than a wrap, to immobilize the mice. The volume of blood that may be obtained from the mouse using venipuncture of the submandibular vascular bundle (200-500 &#181;l) is greater than can be safely collected from the tail vein (200 &#181;l maximum). Because sampling blood from the submandibular vascular bundle requires minimal restraint and may yield more blood, this is the preferred route for sampling in the mouse.
The rapidity with which this procedure may be performed, along with its minimally invasive nature, also minimizes the potential perturbation of blood-based measures by the acute stress response6. The acute stress response can alter circulating levels of many molecules, including interleukins and other immune-active factors7, hormones of the hypothalamic-pituitary-adrenal axis8, hormones in the sympathetic nervous system9, ghrelin10, endogenous opioids11, dopamine, and serotonin12. If resting circulating measures of these molecules or others regulated by these molecules are desired, it is important to minimize the stress response, which is triggered within as little as a minute of the start of stressor exposure.
Stress responses not only alter the composition of the blood, but also represent a technical obstacle for blood sampling because of the constriction of vasculature via increased drive from the sympathetic nervous system. It becomes increasing difficult to obtain steady blood flow from a rat that is mounting an acute stress response. Therefore, the animal&#39;s distress must be minimized in order to rapidly obtain samples that reflect the physiological state of interest.

Biomarkers obtained from blood provide useful diagnostic, predictive, and stratifying measures in many clinical contexts, including cardiovascular disease1, cancer sciences2, and psychiatric disease3. They may also be used in basic science to assess the &#8220;state&#8221; of an organism, including the degree of hunger, inflammation, or stress present. Such measures can be influenced by variables that may or may not be critical to the question of interest, including the time of day that the sample is obtained and the gender of the subjects. It may also be influenced by the stress induced during the blood sampling procedures itself. Stress hormones and the perception of pain can rapidly alter the composition of the blood.
Rodents are the most commonly used laboratory animal, and multiple methods have been developed for blood collection. The ideal method of blood sampling should have minimal physiological impact on the animal, require no anesthesia, allow rapid and repeated sampling, and provide sufficient blood volume for numerous downstream applications. Popular techniques for collecting blood such as catheterization of the jugular vein or tail tip amputation do not meet these criteria.
The aim of this protocol is to describe a blood sampling technique for use in rats that is minimally stressful, does not require anesthesia, allows for multiple blood collections within a single subject, and provides a relatively large sample volume such that multiple assays may be performed on a single sample. The goal of this method is to obtain blood samples that are minimally influenced by the acute stress response.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium heparin (1000 USP units/ml)</td>
			<td>Patternson Veterinary Supply</td>
			<td>25021040010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>JT Taylor</td>
			<td>JT2020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermachlor Rinse-Chlorhexadine</td>
			<td>Butler Schein</td>
			<td>6356</td>
			<td>Topical antiseptic solution, 2% chlorhexidine gluconate</td>
		</tr>
		<tr>
			<td>SURFLO Winged Infusion Sets, Terumo, butterfly catheters</td>
			<td>VWR Scientific</td>
			<td>TESV25BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Tuberculin 1cc syringes</td>
			<td>VWR Scientific</td>
			<td>BD309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>89202-682</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#956;l microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>21150-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, stainless steel, 5&#34;</td>
			<td>VWR Scientific</td>
			<td>82027-586</td>
			<td></td>
		</tr>
		<tr>
			<td>500ml plastic beaker</td>
			<td>VWR Scientific</td>
			<td>414004-149</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean cloth wrap</td>
			<td>Butler Schein</td>
			<td>2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Velcro tape, .75&#34; width</td>
			<td>Monoprice</td>
			<td>B004AF9II6</td>
			<td>Hook and loop tape</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>VWR Scientific</td>
			<td>62344-641</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 plasma collected from the lateral tail vein as described in the protocol gives a plasma sample that was translucent and pale yellow in appearance. As shown in Figure 1, hemolysis in a sample imparts a red tint to the plasma. The acute stress response can rapidly alter the composition of blood. For example, circulating corticosterone concentration can markedly increase within 10 min of stressor exposure, as shown in Figure 2. The low basal levels of corticosterone obtained with this method prior to stressor exposure reveal that the sampling procedure itself is not a significant source of stress.
<img alt="Figure 1" src="/files/ftp_upload/52766/52766fig1.jpg" /> 
Figure 1: Sample appearance. (A) A hemolyzed sample is shown. After centrifugation, the plasma or serum layer (surface indicated by the black arrow) appears tinged with pink or red. Darker tints indicate greater levels of hemolysis. (B) After centrifugation, a properly collected sample will have a clear, yellowish appearance to the upper band (surface indicated by the black arrow), which corresponds to the non-hemolyzed plasma or serum. When removing this layer, it is important to not disturb the underlying whole blood, either by pushing the pipette tip into the whole blood layer or by aspirating some of the whole blood into the tip. Any plasma or serum contaminated with whole blood should be discarded.
<img alt="Figure 2" src="/files/ftp_upload/52766/52766fig2.jpg" /> 
Figure 2: Plasma corticosterone is rapidly elevated following a stressful experience. Blood was obtained from the lateral tail vein of adult female Long-Evans rats before and 10 min following exposure to 4 tones (10 sec, 2 kHz, 85 dB) co-terminating with footshocks (1 sec, 350 &#181;A). Blood plasma corticosterone at baseline (290.4 &#177; 138.8 pg / ml) was significantly less than the levels observed 10 min following presentation of the footshock stress (2204.8 &#177; 454.5 pg / ml, p = 0.02, n = 4), as determined by paired t-test. *, p &#60;0.05

Watch this Scientific Journal Video about Sampling Blood from the Lateral Tail Vein of the Rat at JoVE.com

Sampling Blood from the Lateral Tail Vein of the Rat

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, ...

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102.1K Views.  Massachusetts Institute of Technology. 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.

Video: Sampling Blood from the Lateral Tail Vein of the Rat

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

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 ...

Methods Article

Sampling Blood from the Lateral Tail Vein of the Rat