This work was supported by the National Nature Science Foundation of China (81570399 and 81773735), the National Key Research and Development Program of China - Traditional Chinese Medicine Modernization Research project (2017YFC1702003), and Hei Long Jiang Outstanding Youth Science Fund (JC2017020).

All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
NOTE: The tools and equipment required for the method are listed in the Table of Materials.
1. Preparation of materials and animals
<ol>
	<li>Order C57BL/6 male mice with weight between 20 g-25 g from a standard laboratory animal supplier.</li>
	<li>House the mice in a cycle of 12 h light: 12 h darkness at 24&#8722;26 &#176;C with ad libitum access to water and food.</li>
</ol>
2. Parabiosis
<ol>
	<li>Anesthetize the mice by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol at a concentration of 0.1 mL/10 g. Confirm proper anesthetization as indicated by muscle relaxation, slow and steady breathing, loss of skin stimulation reflex, and disappearance of corneal reflex.</li>
	<li>Apply ophthalmic ointment with a Q-tip to prevent dry eyes.</li>
	<li>Perform the parabiosis procedure as previously described5.
	<ol>
		<li>Place the mice in the supine position. Thoroughly shave the left side of one mouse and the right side of the other mouse starting at approximately 1 cm above the elbow to 1 cm below the knee with an electric shaver. Use depilatory cream to totally clear the fur on the shaved skin.</li>
		<li>Wipe the shaved areas with iodophor. Place the mice on a heated pad covered by a sterile pad.</li>
		<li>Create longitudinal skin incisions starting from 0.5 cm above the elbow to 0.5 cm below the knee joint using a pair of sharp scissors on each animal&#39;s shaved side.</li>
		<li>Detach the skin from the subcutaneous fascia gently following the incision.</li>
		<li>Connect the olecranon and knee of the parabiont with a 3-0 suture.</li>
		<li>Suture the shaved skin with a continuous 5-0 suture.</li>
	</ol>
	</li>
	<li>Inject 0.5 mL of 0.9% NaCl subcutaneously to each mouse to prevent dehydration.</li>
	<li>Inject tramadol (10 mg/25 g/day) intramuscularly to each mouse to relieve pain.</li>
</ol>
3. Validation of circulation chimerism
<ol>
	<li>Glucose fluctuation method 
	NOTE: Verify the successful construction of circulation chimerism between parabionts using the glucose fluctuation method (no fasting) on the 10th day after parabiosis surgery.
	<ol>
		<li>Anesthetize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.1 mL/10 g.</li>
		<li>Fix the donor mice in a Venous visual-mouse tail fixator.</li>
		<li>Rub the caudal veins in the flank of one of the parabiont&#39;s tail with a cotton ball soaked in 70&#8722;75% alcohol to clean the tail and dilate the blood vessels.</li>
		<li>Hold a 1.0 mL syringe containing glucose in the right hand and keep the needle parallel to the vein (less than 15&#176;).</li>
		<li>Insert the needle at a position approximately 2&#8722;4 cm from the tail tip.</li>
		<li>Inject 100 &#181;L of glucose (1.2 g/kg) into the donor within 10 s. 
		NOTE: The term donor refers to the parabiont that receives the glucose injection through the tail vein. The recipient is the other parabiont, which does not receive glucose directly.</li>
		<li>Clean the toes with iodophor. Cut off the toes of the donor and recipient mice with a scissor and collect a drop of blood at different time points after the injection of glucose (1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, and 60 min). Remove the blood clot at each collection time point to avoid more damage. 
		NOTE: The volume of collected blood was 10-25 ul for one test, and 90-225 ul in total.</li>
		<li>Drip the blood into the center of glucometer test strips for glucose level detection.</li>
	</ol>
	</li>
	<li>Use Evan&#39;s blue Counterstain as a positive control3.
	<ol>
		<li>Inject 200 &#181;L of 0.5% Evan&#39;s blue Counterstain intraperitoneally into the donor mouse.</li>
		<li>2 h later, euthanize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.2 mL/10 g, and collect blood from both parabionts by cardiac puncture.</li>
		<li>Centrifuge the blood samples at 916 x g for 15 min.</li>
		<li>Collect serum from the supernatant.</li>
		<li>Dilute the serum with 0.9% NaCl at 1:50.</li>
		<li>Measure the absorbance of the diluted serum samples at 620 nm with a spectrophotometer.</li>
	</ol>
	</li>
</ol>

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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=Growth+differentiation+factor+11+is+a+circulating+factor+that+reverses+age-related+cardiac+hypertrophy.">Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.</a> Cell. 153 (4), 828-839 (2013).</li><li>Kamran, 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=Parabiosis+in+mice:+a+detailed+protocol.">Parabiosis in mice: a detailed protocol.</a> Journal of Visualized Experiments. (80), e50556 (2013).</li><li>Conboy, M. J., Conboy, I. M., Rando, T. 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The authors have nothing to disclose.

Parabiosis refers to the surgical technique of connecting two living animals to establish a common vascular system by experimental means6,7,8. The advantage of this model is that the substances produced by a single individual can act on both animals at the same time. Thus, the parabiosis model can be used to explore the role of a substance or factor in a related disease, producing many meaningful and innovative conclusions. In view of its great application value, the model has brought about a better understanding of cardiovascular system diseases8,9,10,11,12,13, nervous system disorders14,15, organ transplantation16, and diabetes17,18.
However, verification of the successful establishment of circulation chimerism is the first and determining step for studies with parabiosis. Current studies have reported some methods for detecting circulation chimerism. Loffredo et al.4 reported that blood chimerism was confirmed in parabiotic pairs by measuring the mixed frequency of monocytes in the spleen with different labeled markers in the donor (CD45.1+) and recipient (CD45.2+) mice. In this method, specific mice with CD45.1+- or CD45.2+-labeled monocytes for each parabiont were used for parabiosis. Cell cytometry was needed to determine blood chimerism by measuring the integration of the marked blood cells. In addition, Marta et al.3 assessed cross-circulation by intraperitoneally injecting 200 &#181;L of 0.5% Evans blue dye in one of the parabionts. Blood from both parabionts was collected 2 h later by cardiac puncture. Blood chimerism was determined by an increased Evans blue concentration in the recipient mice, which was tested by a microplate reader. Although these strategies allow us to determine blood chimerism, there are still many limitations that cannot be ignored. First, for lethal methods, the blood chimerism can only be confirmed at execution. However, in our method, the establishment of blood chimerism can be tested any time after parabiosis surgery. Parabionts with unsuccessful established circulation chimerism can be excluded for further study in advance to reduce unnecessary workload. Indeed, using our glucose fluctuation method we were able to pick out the mice with unsuccessful construction of blood chimerism in certain parabionts, which might be due to insufficient time of parabiosis, unstable surgery manipulation, delayed wound healing, or disconnected body tissue caused by animal struggling. In such circumstances, the failure of blood chimerism was also confirmed by using the Evans blue method. These results indicate that the glucose fluctuation method was as effective as the Evans blue method (Supplementary Figure 1 and Supplementary Figure 2, Supplementary Table 1 and Supplementary Table 2). Second, the currently used methods of validation are excessively time-consuming and difficult, as opposed to this new method, which is simple and effective.
In the present study, we successfully tested for circulation chimerism in parabiotic mice by the glucose fluctuation method. The mice were anesthetized for the glucose injection and blood glucose level measurements, which made their manipulation easier. We injected 100 &#181;L of glucose (1.2 g/kg) through the tail vein of the mice within 10 s. Under these conditions, a regular fluctuation of blood glucose levels in donor and recipient mice was observed. Importantly, the amount of glucose we used tended to stably elevate the blood glucose level within a certain range. Moreover, the results showed that the blood insulin level recovered to the normal range 3 h after glucose injection (Supplementary Figure 3), suggesting that the amount of glucose injected into the mice had minimal effects on insulin metabolism. In addition, the dosage of glucose we gave to the donor was lower than that used for the glucose tolerance test of mice (for the GTT, 2 g/kg glucose is intraperitoneally injected to the mice, equaling approximately 1.6 g/kg through caudal vein injection19,20,21), which means that the dosage of glucose for the glucose fluctuation method would not cause hyperglycemia and other damaging alterations. In conclusion, the method we used is effective, harmless, and timesaving. However, it might be limited to diabetic mice in parabiosis, which still need further experiments for confirmation.

Parabiosis is a modeling method in which two living organisms are joined together surgically and develop as a single physiological system with a shared circulatory system1. Such models have been widely used for studying physiology owing to the advantage that the substances produced by a single individual can act on both animals at the same time via the shared circulatory system. Since the mid-1800s when parabiotic experiments were pioneered by Paul Bert2, the methods for constructing parabiotic models have become standardized. However, a straightforward and convenient method for verifying the successful establishment of blood chimerism has been missing. It has been reported that cross-circulation can be successfully assessed by intraperitoneally injecting 0.5% Evans blue dye in one of the parabionts followed by measurement of the absorbance of Evans blue in the blood of both parabionts with a microplate reader3. Another method requires a specific mouse breed that contains CD45.1+- and CD45.2+-labeled monocytes in each parabiont. Cell cytometry is then used to determine blood chimerism by measuring the frequency of the two markers in monocytes from spleen or blood4. However, these methods are often lethal or cumbersome to the animals, and a safe and simple method for quick and reliable verification of parabiotic models is highly desirable. In this study, we established a new method for this purpose, which was validated in a mouse model of parabiosis. Glucose concentration in blood samples drawn from a tail vein is measured using a glucometer, and the pattern of changes of glucose level in donor and recipient mice is considered an indication of circulation chimerism. We named this method the &#34;glucose fluctuation method&#34;. The application of this validation method is not limited to mice but could be extended to diverse pathological models except for those with serious dysregulation of glucose metabolism. The procedure is simple, timesaving, and safe.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>curved forceps</td>
			<td>JZ surgical Instruments, China</td>
			<td>J31340</td>
			<td></td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>JZ surgical Instruments, China</td>
			<td>WA1030</td>
			<td></td>
		</tr>
		<tr>
			<td>needle forcep</td>
			<td>JZ surgical Instruments, China</td>
			<td>60017961</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringes</td>
			<td>Agilent, USA</td>
			<td>5182-9642</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethano</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T48402-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Solarbio, China</td>
			<td>IP0150</td>
			<td></td>
		</tr>
		<tr>
			<td>tramadol</td>
			<td>Yijishiye, China</td>
			<td>YJT712520</td>
			<td></td>
		</tr>
		<tr>
			<td>glucometer</td>
			<td>Roche Diabetes Care, Indiana</td>
			<td>Accu-Chek Active test strips</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#39;s blue Counterstain</td>
			<td>Solarbio, China</td>
			<td>G1810</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Nair, USA</td>
			<td>LL9161</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp, USA</td>
			<td>TP-22G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Codos, China</td>
			<td>CP-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0,5-0 
			surgical suture</td>
			<td>Shanghai Medical Suture Needle Factory, China</td>
			<td>SYZ 3-0#, SYZ 5-0#</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In six donor mice, blood glucose levels sharply increased to 26.5 &#181;mol/L (173% increase) at an average of 1 min after the injection of 100 &#181;L of glucose (1.2 g/kg) through the tail vein and then gradually decreased to 13.3 &#181;mol/L at 60 min. In recipient mice, blood glucose slowly increased after injection and reached the first peak level at 15 min (47% increase, 12.2 &#181;mol/L). Based on the above results, the standard for circulation chimerism was set as follows: 1) a sharp increase in the blood glucose level (a minimum 100% increase or &#62;20 &#181;mol/L) in donor mice within 1 min after glucose injection, and 2) a significant increase in the blood glucose level in recipient mice 15 min after injection (a minimum 37% increase) (Figure 1).
The concentration of Evans blue dye in the serum of parabionts also indicated the successful construction of circulation chimerism (Figure 2). We euthanized the parabionts after blood glucose measurement using 5 mL of 2,2,2-tribromoethanol (20 g/L). The subcutaneous vascular junctions between the parabionts were clearly observed (Figure 3).
Supplementary Figure 1 shows that the donor mice had a significant blood glucose level increase 1 min after glucose injection, while the blood glucose level of the recipient mice was not elevated, which demonstrated that the circulation chimerism in parabionts was not successfully established 1 day after parabiosis surgery. Similarly, the blood OD level in recipient mice was not as elevated as that of donor mice (Supplementary Figure 2).
Based on the results from the glucose fluctuation method, we found that two pairs of parabionts did not establish blood chimerism 15 days after parabiosis surgery. As shown in Supplementary Table 1, the two recipient mice did not have an increased blood glucose level within 60 min after glucose injection into the donor mice. The blood concentration of Evans blue in the two recipients was also not elevated (Supplementary Table 2), which demonstrated that the glucose fluctuation method was as sensitive as the Evans blue method.
Moreover, to evaluate the influence of injected glucose on insulin metabolism, we detected the blood insulin level 1 h and 3 h after injection of 100 &#181;L of glucose (1.2 g/kg) in mice (Supplementary Figure 3). The blood insulin level was remarkably decreased 1 h after glucose injection because of quickly increased glucose and recovered to normal levels at 3 h. These results demonstrated that the effects of glucose we injected on insulin metabolism were restorable.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60411/60411fig1.jpg" /> 
Figure 1: Changes of blood glucose level in parabionts after injection of glucose through the caudal vein. (A) Blood glucose level of donor mice. (B) Blood glucose level of recipient mice (n = 6). The data are presented as the mean &#177; SEM. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 vs. 0 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60411/60411fig2.jpg" /> 
Figure 2: The concentration of Evans blue in serum samples of parabionts measured by a microplate reader. The data are presented as the mean &#177; SEM. ***p &#60; 0.001 (n = 6). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60411/60411fig3.jpg" /> 
Figure 3: Generation of subcutaneous vasoganglions in connected skin between the parabionts. Left: Representative image of the parabiosis mice. Right: A representative image of the subcutaneous vasoganglion between the parabionts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60411/60411suppfig1.jpg" /> 
Supplementary Figure 1: The blood glucose level of parabionts was tested 1 day after parabiosis surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411suppfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/60411/60411suppfig2.jpg" /> 
Supplementary Figure 2: The concentration of Evans blue measured by microplate reader in the serum of the parabionts 1 day after parabiosis surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411suppfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 3" src="/files/ftp_upload/60411/60411suppfig3.jpg" /> 
Supplementary Figure 3: The concentration of insulin in the serum of the mice after glucose injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60411/60411suppfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>0 min</td>
			<td>5 min</td>
			<td>15 min</td>
			<td>20 min</td>
			<td>40 min</td>
			<td>60 min</td>
		</tr>
		<tr>
			<td>Donor-1</td>
			<td>5.7</td>
			<td>26.2</td>
			<td>21.2</td>
			<td>17.6</td>
			<td>16.9</td>
			<td>15.4</td>
		</tr>
		<tr>
			<td>Recipient-1</td>
			<td>6.7</td>
			<td>5.8</td>
			<td>5.9</td>
			<td>6.2</td>
			<td>5.2</td>
			<td>5.4</td>
		</tr>
		<tr>
			<td>Donor-2</td>
			<td>8.4</td>
			<td>25.5</td>
			<td>21.1</td>
			<td>20.5</td>
			<td>17.4</td>
			<td>13.8</td>
		</tr>
		<tr>
			<td>Recipient-2</td>
			<td>6.7</td>
			<td>5.8</td>
			<td>5.9</td>
			<td>6.2</td>
			<td>5.2</td>
			<td>5.4</td>
		</tr>
	</tbody>
</table>
Supplementary Table 1: Blood glucose level (&#181;mol/L) in parabionts 15 days after parabiosis surgery.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Control-1</td>
			<td>Donor-1</td>
			<td>Recipient-1</td>
			<td>Control-2</td>
			<td>Donor-2</td>
			<td>Recipient-2</td>
		</tr>
		<tr>
			<td>OD value</td>
			<td>0.059</td>
			<td>0.935</td>
			<td>0.062</td>
			<td>0.068</td>
			<td>0.862</td>
			<td>0.073</td>
		</tr>
	</tbody>
</table>
Supplementary Table 2: Blood concentration of Evans blue (OD value) in parabionts 15 days after parabiosis surgery.

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Biology

8.3K Views.  Harbin Medical University. 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.

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

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: ...

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 ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

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

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Chapters in this video

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

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Sampling Blood from the Lateral Tail Vein of the Rat

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Activating Autophagy by Aerobic Exercise in Mice

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Detecting Migration and Infiltration of Neutrophils in Mice