Name: Cardiac Blood Collection via Subclavian Puncture in Mice (Video) | JoVE Journal
Uploaded: 2025-11-28T14:25:00.000+00:00
Duration: 4 min 47 s
Description: Learn subclavian cardiac puncture for high-purity blood collection in mice using C57BL/6 models. This method avoids liver damage and yields 700-1,200 microliters reliably.

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5 minutes

This work was funded by grants from the National Natural Science Foundation of China (No. 82360065) and the Natural Science Foundation of Jilin Province (Grant No. YDZJ202501ZYTS166). The authors thank Gang Chen from Yanbian University for help with the cardiac blood sampling methods.

All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. The study strictly adhered to ethical regulations for animal experiments and was approved by the Animal Ethics Committee (Approval Number: YD20250310007). A total of 14 healthy C57BL/6 mice (seven males and seven females) were used in this study, with body weights ranging from 21 g to 29 g. The animals were housed at the Laboratory Animal Center of Yanbian University under standard laboratory conditions.
1. Animals
<ol>
	<li>Following terminal cardiac blood collection, immediately euthanize mice by overdose of isoflurane in an induction chamber (5%) until respiratory arrest and loss of nociceptive reflexes are observed. Confirm death by absence of spontaneous respiration and cardiac activity. 
	NOTE: Handle needles with caution to prevent needlestick injury, and immediately discard all used sharps into designated sharps containers. Dispose of biological waste and animal carcasses in designated biohazard containers.</li>
</ol>
2. Experimental preparation
<ol>
	<li>Get the following materials ready for the experiment: a 2 mL syringe with a 25 G needle, 75% ethanol, 1.5 mL microcentrifuge tubes with a tube rack, heparin, a low-temperature ice box, an electronic scale, a 4 &#176;C centrifuge, a pipette, and pipette tips.</li>
	<li>For cardiac blood collection in mice, use a 2 mL syringe due to its optimal volume, stable control, enhanced operational flexibility, and reduced risk of blood coagulation.</li>
	<li>Rinse the syringe with heparin solution (0.3 mL, 1,250 U/mL) to prevent blood clotting.</li>
</ol>
3. Mouse preparation and positioning
<ol>
	<li>Select healthy mice and weigh them.</li>
	<li>Administer inhalation anesthesia using isoflurane. Place the mouse in an induction chamber and expose it to 5% isoflurane gas until fully anesthetized. Confirm anesthesia when the following criteria are met: loss of the righting reflex, loss of the pain reflex.</li>
	<li>Monitor respiration, color, and reflexes throughout the procedure to ensure adequate anesthesia.</li>
</ol>
4. Puncture procedure
<ol>
	<li>Place the mouse in a supine position and securely fix it on the operating platform with its forelimbs stabilized (Figure 1). Disinfect the thoracic area with 75% ethanol, ensuring proper identification of the xiphoid process and left clavicle (Figure 2). 
	NOTE: When applying ethanol, avoid contact with the mouse&#39;s mouth and nose to prevent inhalation of alcohol vapors, which may cause respiratory distress.</li>
	<li>Hold the syringe with the needle pointing downward. 
	NOTE: The primary reason for this orientation is to enhance operational stability when the mouse is fixed in a supine position, reducing needle movement and sampling error during blood collection.</li>
	<li>Insert the 2 mL syringe from the midline below the left clavicle, directing the needle toward the xiphoid process (Figure 2 and Figure 3).</li>
	<li>Advance the needle to a depth of 1-1.5 cm while keeping the syringe parallel to the operating table.</li>
	<li>Confirm the correct positioning of the needle within the heart chamber by observing a pulsatile blood return into the syringe. 
	NOTE: The operator should maintain a steady hand to prevent the needle from shifting or penetrating too deeply.</li>
</ol>
5. Blood collection
<ol>
	<li>Once the needle is correctly positioned, slowly draw back the syringe plunger to collect blood. Maintain a steady hand throughout the blood collection process to prevent needle movement, which could disrupt blood flow. 
	NOTE: If blood flow is insufficient, slight adjustments to the syringe angle may help improve collection. However, avoid frequent repositioning of the needle to minimize experimental errors and reduce the risk of tissue damage.</li>
</ol>
6. Sample processing
<ol>
	<li>Immediately transfer the blood sample into a pre-heparinized tube to prevent coagulation and preserve sample integrity.</li>
	<li>Centrifuge the blood sample at 4 &#176;C for 15 min at 2,000 &#215; g to effectively separate plasma from blood cells.</li>
	<li>Following centrifugation, examine the sample for clear separation of plasma and blood cells (Figure 4A). Record the sample condition to ensure it meets experimental requirements (Figure 4B).</li>
	<li>Using a pipette, carefully extract the supernatant (plasma) and transfer it to a new tube. Label the tube appropriately for subsequent analysis or storage.</li>
</ol>
7. Sample testing
<ol>
	<li>Analyze the prepared plasma samples for AST and ALT levels using an ELISA assay kit.</li>
</ol>

<ol><li>Jo, E. J., Bae, E., Yoon, J. H., Kim, J. Y., Han, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+murine+retroorbital+plexus+and+facial+vein+blood+collection+to+mitigate+animal+ethics+issues%5BTitle%5D)+AND+%22Lab+Anim+Res%22%5BJournal%5D)">Comparison of murine retroorbital plexus and facial vein blood collection to mitigate animal ethics issues.</a> Lab Anim Res. 37 (1), 12(2021).</li>
<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Best+practices+for+blood+collection+and+anaesthesia+in+mice%3A+selection%2C+application+and+reporting%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">Best practices for blood collection and anaesthesia in mice: selection, application and reporting.</a> Br J Pharmacol. 182 (11), 2337-2353 (2025).</li>
<li>Fernández, I., Peña, A., Del Teso, N., Pérez, V., Rodríguez-Cuesta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+biochemistry+parameters+in+C57BL%2F6J+mice+after+blood+collection+from+the+submandibular+vein+and+retroorbital+plexus%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus.</a> J Am Assoc Lab Anim Sci. 49 (2), 202-206 (2010).</li>
<li>Kaur, H., Fisher, K., Othman, M. J. B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thromboelastography+testing+in+mice+following+blood+collection+from+facial+vein+and+cardiac+puncture%5BTitle%5D)+AND+%22Blood+Coagul+Fibrinolysis%22%5BJournal%5D)">Thromboelastography testing in mice following blood collection from facial vein and cardiac puncture.</a> Blood Coagul Fibrinolysis. 30 (7), 366-369 (2019).</li>
<li>Beeton, C., Garcia, A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drawing+blood+from+rats+through+the+saphenous+vein+and+by+cardiac+puncture%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Drawing blood from rats through the saphenous vein and by cardiac puncture.</a> J Vis Exp. (7), e266(2007).</li>
<li>Doeing, D. C., Borowicz, J. L., Crockett, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+dimorphism+in+differential+peripheral+blood+leukocyte+counts+in+mice+using+cardiac%2C+tail%2C+foot%2C+and+saphenous+vein+puncture+methods%5BTitle%5D)+AND+%22BMC+Clin+Pathol%22%5BJournal%5D)">Gender dimorphism in differential peripheral blood leukocyte counts in mice using cardiac, tail, foot, and saphenous vein puncture methods.</a> BMC Clin Pathol. 3 (1), 3(2003).</li>
<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sample+collection+in+small+laboratory+animals%5BTitle%5D)+AND+%22J+Pharmacol+Pharmacother%22%5BJournal%5D)">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87(2010).</li>
<li>Cao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver-heart+cross-talk+mediated+by+coagulation+factor+XI+protects+against+heart+failure%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Liver-heart cross-talk mediated by coagulation factor XI protects against heart failure.</a> Science. 377 (6613), 1399-1406 (2022).</li>
<li>Ward, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GWAS+of+serum+ALT+and+AST+reveals+an+association+of+SLC30A10+Thr95Ile+with+hypermanganesemia+symptoms%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">GWAS of serum ALT and AST reveals an association of SLC30A10 Thr95Ile with hypermanganesemia symptoms.</a> Nat Commun. 12 (1), 4571(2021).</li>
<li>Hu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+inferior+vena+cava+puncture+under+continuous+cardiac+perfusion+with+cardiac+puncture+in+blood+acquisition+of+the+laboratory+mouse%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Comparison of inferior vena cava puncture under continuous cardiac perfusion with cardiac puncture in blood acquisition of the laboratory mouse.</a> Lab Anim. 59 (2), 178-191 (2025).</li>
<li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarwood+extract+mitigates+alcoholic+fatty+liver+in+C57+mice+via+anti-oxidation+and+anti-inflammation%5BTitle%5D)+AND+%22Mol+Med+Rep%22%5BJournal%5D)">Agarwood extract mitigates alcoholic fatty liver in C57 mice via anti-oxidation and anti-inflammation.</a> Mol Med Rep. 28 (5), 210(2023).</li>
<li>Ansere, V. A., Bubak, M. P., Miller, B. F., Freeman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochronic+plasma+transfer%3A+experimental+design%2C+considerations%2C+and+technical+challenges%5BTitle%5D)+AND+%22Rejuvenation+Res%22%5BJournal%5D)">Heterochronic plasma transfer: experimental design, considerations, and technical challenges.</a> Rejuvenation Res. 26 (5), 171-179 (2023).</li>
<li>Kress, A. P. A., Zhang, Y., Kaiser-Vry, A. R., Sauer, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+blood+collection+techniques+in+mice+and+their+effects+on+welfare%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">A comparison of blood collection techniques in mice and their effects on welfare.</a> J Am Assoc Lab Anim Sci. 61 (3), 287-295 (2022).</li>
<li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemolysis+control+in+the+emergency+department+by+interventional+blood+sampling%5BTitle%5D)+AND+%22J+Pers+Med%22%5BJournal%5D)">Hemolysis control in the emergency department by interventional blood sampling.</a> J Pers Med. 13 (4), 651(2023).</li>
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The authors have no conflicts of interest to disclose.

Cardiac puncture is a widely used terminal blood collection method in mouse experiments, ideal for obtaining large volumes of blood while minimizing contamination from tissue fluids, thus ensuring sample purity12. Compared to other blood collection techniques, cardiac puncture is straightforward, requiring only a single procedure, which helps reduce stress in experimental animals. In this study, we employed the subclavian puncture method. This approach features a well-defined needle path, minimizi...

Blood collection from mice is essential in scientific research and various other fields1. Non-terminal blood collection methods, including tail vein, femoral vein, and submandibular sampling, provide advantages in repeatability, yet they carry potential risks of tissue damage, animal stress, and compromised sample quality2. In terminal procedures, cardiac puncture and large-vessel blood collection are commonly used to obtain large blood samples, typically yielding approximately 50% of the total blood volume3,4.
Large-vessel blood collection requires a surgical incision and carries a high bleeding risk. In contrast, cardiac puncture is minimally invasive, reduces animal stress, and is relatively simple to perform5. By directly collecting blood from the heart, this method ensures a swift blood flow, minimizing the risk of coagulation and improving sample quality6. As a result, cardiac puncture is the preferred technique for terminal experiments7.
In this study, we employed the subclavian approach for cardiac puncture. Compared with traditional puncture methods, this approach offers a more precise needle trajectory. The needle sequentially traverses the skin, subcutaneous tissue, and thoracic muscles, enters the thoracic cavity, and reaches the cardiac chamber for blood collection. Abdominal puncture, for instance, may cause liver injury, which can alter blood components such as serum proteins, coagulation factors, and metabolic enzymes8. 
Additionally, liver damage can lead to elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, further interfering with experimental results9. In practice, the subclavian approach is particularly suitable for experiments that require intact liver tissue, precise assessment of hepatic function, or sensitive measurement of proteins and metabolites. However, it is less suitable for very young or small mice, where anatomical structures are difficult to visualize, and for studies requiring intact cardiac tissue, as puncture may compromise subsequent analyses.

<table><tbody><tr><td>1.5 mL Tubes</td><td>Labselect</td><td>MCT-001-150-S</td><td></td></tr><tr><td>15 mL Conical Tubes&amp;nbsp;</td><td>As One Corp</td><td>1-3500-01&amp;nbsp;</td><td></td></tr><tr><td>2 mL Syringe</td><td>Jiangxi Fenglin</td><td>0.5x20RWLB</td><td></td></tr><tr><td>4 &amp;deg;C Centrifuge</td><td>Eppendorf</td><td>5409CP202285</td><td></td></tr><tr><td>75% Ethanol</td><td>Nanjing Reagent</td><td>C069156931</td><td></td></tr><tr><td>Alanine Aminotransferase (ALT/GPT) Activity Assay Kit</td><td>Elabscience&amp;reg;&amp;nbsp;</td><td>E-BC-K235-M</td><td></td></tr><tr><td>Aspartate Aminotransferase (AST/GOT) Activity Assay Kit</td><td>Elabscience&amp;reg;&amp;nbsp;</td><td>E-BC-K236-M</td><td></td></tr><tr><td>Electronic Compact Scale</td><td>YUHENG</td><td>YH-400A</td><td></td></tr><tr><td>Heparin</td><td>solarbio</td><td>H8060</td><td></td></tr><tr><td>Inhalation Anesthesia Machine</td><td>Mindray</td><td>10-1020</td><td></td></tr><tr><td>&amp;nbsp;Isoflurane</td><td>RWD Life Science</td><td>R510-22-16</td><td></td></tr><tr><td>Pipette</td><td>Eppendorf</td><td>3111 000 840</td><td></td></tr><tr><td>Pipette tips</td><td>&amp;nbsp;Axygen&amp;reg;&amp;nbsp;&amp;nbsp;</td><td>MTX-1250-C</td><td></td></tr></tbody></table>

cardiac blood collection via subclavian puncture in mice

Cardiac Blood Collection via Subclavian Puncture in Mice

In experimental studies, mouse blood is a common experimental sample. However, there are still some challenges for the collection of large volumes of blood. The common methods of terminal large-volume blood collection include large-vessel blood collection and cardiac puncture collection. Large-vessel blood collection often requires surgery and is difficult to perform. In contrast, cardiac puncture is simple and easy to operate. However, there are also different methods for blood sampling by cardiac puncture. Different methods have different puncture sites, and each has its advantages and disadvantages. In this study, we provided a detailed examination of the subclavian cardiac puncture technique for blood collection, emphasizing its ease of operation and its ability to yield high-quality samples. This study validates that the subclavian cardiac puncture blood collection method can recover approximately 50% of the total blood volume and significantly reduce tissue fluid contamination. The method can be applied to multiple research disciplines, including immunology, toxicology, and metabolic studies.

In this study, we conducted repeated experiments on 14 C57BL/6 mice (21-29 g), with seven males and seven females. Studies report that the estimated blood volume in an adult mouse is 55 to 70 mL/kg body weight10. The blood volume collected per session ranged from 700 to 1,200 µL, accounting for approximately 50% of the total circulating blood volume (Figure 5). The operation time per session ranged from 60 to 80 s. No hemolysis or sample conta...

Subclavian cardiac puncture is a simple, reliable method for collecting terminal blood in mice. It is easy to operate and ensures high-purity samples.

In experimental studies, mouse blood is a common experimental sample. However, there are still some challenges for the collection of large volumes of ...

Cardiac Blood Collection via Subclavian Puncture in Mice. Blood sampling in mice plays a significant role in scientific research. Cardiac puncture and large-vessel blood collection can obtain large blood volumes in terminal procedures.However, cardiac puncture has received increasing attention due to ethical limitations and higher associated risks. In this study, we employed the subclavian puncture method for cardiac blood collection. Compared with the traditional abdominal puncture approach, this method effectively avoids liver damage, thereby improving sample quality and the reliability of experimental data.When drawing blood, the syringe needle should be facing downward. Insert the needle from the left midclavicular line direction, aiming the needle towards the xiphoid process. Experiment preparation.In intracardiac blood collection in mice, the one-milliliter syringe is prone to needle dislodgement due to its thin barrel and long plunger stroke. The two-milliliter syringe offers better handling than the five-milliliter syringe, making it the optimal choice for this procedure. Use heparin solution to rinse the syringe to prevent blood clotting.Select healthy mice and weigh them. Administer inhalation anesthesia. Then, confirm that the mice have reached an adequate level of anesthesia.Steps for cardiac blood collection in mice. After the mice were anesthetized, they were laid flat on the operating table. The chest area was sterilized with 75%ethanol.Immobilize its forelimbs and wipe off excess alcohol. Hold the syringe with the needle facing downward. Locate the xiphoid process and the left clavicle.Insert the two-milliliter syringe from the left clavicular midline with the needle directed toward the xiphoid process, ensuring the syringe remains parallel to the operating table. Control the insertion depth to 1 to 1.5 centimeters, ensuring that the syringe remains parallel to the operating table at all times. After confirming the correct positioning of the needle, slowly retract the syringe plunger to draw blood.When the needle successfully enters the heart chamber, you will observe pulsatile blood flow into the syringe. During the blood-collection process, hand stability to prevent needle displacement. If difficulties in blood collection occur, adjust the syringe angle as needed to optimize blood flow and increase the success rate.After blood collection, transfer the blood to heparin-coated microcentrifuge tubes. Centrifuge at four degrees Celsius and 2, 000 xg for 15 minutes. Carefully use a pipette to transfer the supernatant plasma to new microcentrifuge tubes and label appropriately.In this study, we conducted repeated experiments on 14 C57BL/6 mice weighing between 21 and 29 grams. Each blood-collection volume ranged from 700 to 1, 200 microliter. All the experimental results are as follows, and no significant differences were observed between the genders.Then, we measured the levels of ALT and AST in the plasma, and the results were all within the normal range. Cardiac puncture offers distinct advantages. Blood is directly drawn from the heart, which effectively avoids contamination from tissue, fluids, or other sources.It requires only a single operation, reducing the stress response caused by repeated sampling in experimental animals. The subclavian puncture method features a well-defined needle trajectory and avoids penetrating the diaphragm or other complex anatomical layers. It also prevents liver damage, thus minimizing alterations in serum proteins, coagulation factors, and metabolic enzyme levels.

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Learn subclavian cardiac puncture for high-purity blood collection in mice using C57BL/6 models. This method avoids liver damage and yields 700-1,200 microliters reliably.

Cardiac Blood Collection via Subclavian Puncture in Mice

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