Name: Facial Vein Venipuncture for Murine Blood Collection (Video) | JoVE Journal
Uploaded: 2025-09-26T14:20:00.000+00:00
Duration: 5 min 1 s
Description: Learn facial vein venipuncture for quick, ethical murine blood collection using a lancet. This simplified method reduces time and skill requirements while enabling serum cytokine analysis and diabetes research.

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5 minutes

This work was funded by the National Institutes of Health Grant: R35 HL155458 (C.V.J.)

Mice were housed in a specific pathogen-free environment at Dartmouth Hitchcock Medical College, an AAALAC-accredited institution, and used in accordance with an approved protocol under the Institutional Animal Care and Utilization Committee.
1. Materials gathering
<ol>
	<li>Prepare 5 mL culture tubes, each containing 1.5 mL of 1x Red Blood Cell (RBC) Lysis Buffer (10X RBC Lysis diluted with sterile water). Use one tube per mouse.</li>
	<li>Gather a box of 3 mm lancets (one lancet per mouse).</li>
	<li>Gather a box of gauze sponges (one gauze sponge per mouse).</li>
	<li>Gather transfer pipets and a 50 mL conical tube containing 1x HBSS with 0.5 mM EDTA (sufficient for up to 20 mice). 
	NOTE: This buffer is used to stop the lysis reaction. If the buffer is only needed for phenotyping and not further downstream analysis, most neutral buffers with pH 7.2- 7.4 can be used in place of HBSS.</li>
	<li>Bring an ice bucket, additional 1x RBC lysis buffer, extra 5 mL tubes, and transfer pipets.</li>
</ol>
2. Mouse and tube labeling
<ol>
	<li>Label each cage card and mouse with a phenotype identifier that corresponds to labels on each tube to aid in distinguishing which results correlate to which mouse. Commonly used identifiers include different permanent marker colors (e.g., red, blue, green, black) on the tails of mice, which correspond to the markings on the tube. 
	NOTE: Always include a control mouse for phenotyping. For reporter mice, ensure to have a negative reporter control. For transgenic mice, include a wild-type mouse as a control.</li>
	<li>Arrange the labeled tubes in a rack with an ice bucket nearby to store tubes immediately after blood collection.</li>
	<li>Open all the lancets needed for the procedure.</li>
	<li>Open all gauze sponges needed for the procedure.</li>
</ol>
3. Mouse handling and sample collection
<ol>
	<li>Use the dominant hand to grasp the mouse by the tail and place it on a wire feeding rack or any flat surface.</li>
	<li>Use the non-dominant index finger and thumb to scruff the mouse by maneuvering from the tail toward the cervical region.</li>
	<li>Use the remaining fingers to secure the tail, minimizing mouse movement.</li>
	<li>Use the dominant hand to retrieve an opened 3 mm lancet.</li>
	<li>Locate the small hairless spot just beyond the mouth and trace a straight line toward the ear. Stop where the line intersects with the outer corner of the eye (Figure 1).</li>
	<li>Insert the lancet at this intersection to ensure an ample blood supply.</li>
	<li>Use the flat lateral edge of the lancet to collect a single drop of blood.</li>
	<li>Deposit the entire lancet, with the blood sample, into the labeled tube containing RBC lysis buffer.</li>
	<li>Gently agitate the tube to ensure the entire sample is mixed into the buffer. Incomplete mixing may cause clotting and poor data quality.</li>
	<li>Use a Gauze sponge to apply gentle pressure to the puncture site for 5-10 s.</li>
	<li>Return the mouse to its cage.</li>
	<li>Maintain a blood-to-lysis buffer ratio greater than 1:10.</li>
	<li>Repeat the steps in this section for each additional mouse.</li>
</ol>
4. Lysis, preparation, and analysis of the sample
<ol>
	<li>Wait for 7-10 min after the start of lysis, add approximately 2 mL of 1x HBSS with 0.5 mM EDTA (or other media).</li>
	<li>Centrifuge tubes at 400 g for 5 min. Prepare antibody cocktails during this time.</li>
	<li>Discard the supernatant.</li>
	<li>Remove the lancets from the tubes using forceps.</li>
	<li>Add 30 &#181;L of an antibody cocktail&#160;designed to identify the desired cellular populations. 
	NOTE: To phenotype MD4 IgHEL mice, our cocktail consisted of 1 mL of HBSS, 25 &#956;L of IgMa PerCP cy-5.5, and 25 &#956;L of CD19 APC.</li>
	<li>Resuspend cells by vortexing for 2-3 s and incubate for 25-30 min at 4 &#176;C.</li>
	<li>Turn on the flow cytometer and configure the flow plots to the needs of the experiment. 
	NOTE: There is no need to spin the cells post-staining if the goal is phenotyping.</li>
	<li>Add 150-25&#8239;&#181;L of HBSS or other media to each sample using a transfer pipette. If needed, DAPI can be added at a final concentration of 1.&#8239;&#181;g/mL (use 10&#8239;&#181;L of DAPI-containing media).</li>
	<li>Vortex each sample for 2-3 s just before acquisition.</li>
	<li>Acquire data 8,000-10,000 events/s, aiming for 100,000 cells if lysis was effective.</li>
	<li>Analyze the acquired data, illustrated in Figure 2 and Figure 3.</li>
</ol>

<ol><li>Harikrishnan, V. S., Hansen, A. K., Abelson, K. S., Sorensen, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+various+methods+of+blood+sampling+in+mice+and+rats%3A+Effects+on+animal+welfare%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">A comparison of various methods of blood sampling in mice and rats: Effects on animal welfare.</a> Lab Anim. 52 (3), 253-264 (2018).</li>
<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>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid%2C+simple%2C+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet%5BTitle%5D)+AND+%22Lab+Anim+%28NY%29%22%5BJournal%5D)">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34 (9), 39-43 (2005).</li>
<li>Francisco, C. C., Howarth, G. S., Whittaker, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+on+animal+wellbeing+and+sample+quality+of+2+techniques+for+collecting+blood+from+the+facial+vein+of+mice%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Effects on animal wellbeing and sample quality of 2 techniques for collecting blood from the facial vein of mice.</a> J Am Assoc Lab Anim Sci. 54 (1), 76-80 (2015).</li>
<li>Ng, M. S., Ng, A. S., Chan, J., Tung, J. P., Fraser, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+packed+red+blood+cell+storage+duration+on+post-transfusion+clinical+outcomes%3A+a+meta-analysis+and+systematic+review%5BTitle%5D)+AND+%22Intensive+Care+Med%22%5BJournal%5D)">Effects of packed red blood cell storage duration on post-transfusion clinical outcomes: a meta-analysis and systematic review.</a> Intensive Care Med. 41 (12), 2087-2097 (2015).</li>
<li>Hoggatt, J., Hoggatt, A. F., Tate, T. A., Fortman, J., Pelus, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bleeding+the+laboratory+mouse%3A+Not+all+methods+are+equal%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">Bleeding the laboratory mouse: Not all methods are equal.</a> Exp Hematol. 44 (2), 132-137.e1 (2016).</li>
<li>Navarro, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+anesthesia%3A+The+art+and+science%5BTitle%5D)+AND+%22ILAR+J%22%5BJournal%5D)">Mouse anesthesia: The art and science.</a> ILAR J. 62 (1-2), 238-273 (2021).</li>
<li>Ahrens Kress, A. P., 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>Lavin, E. S., Feldman, E. R., Soprano, S. M., Moore, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Venipuncture+site+influences+blood-drop+volume+in+C57BL%2F6+mice%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Venipuncture site influences blood-drop volume in C57BL/6 mice.</a> J Am Assoc Lab Anim Sci. 63 (3), 325-332 (2024).</li>
<li>Forbes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morbidity+and+mortality+rates+associated+with+serial+bleeding+from+the+superficial+temporal+vein+in+mice%5BTitle%5D)+AND+%22Lab+Anim+%28NY%29%22%5BJournal%5D)">Morbidity and mortality rates associated with serial bleeding from the superficial temporal vein in mice.</a> Lab Anim (NY). 39 (8), 236-240 (2010).</li>
<li>Moore, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparing+phlebotomy+by+tail+tip+amputation%2C+facial+vein+puncture%2C+and+tail+vein+incision+in+C57BL%2F6+mice+by+using+physiologic+and+behavioral+metrics+of+pain+and+distress%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Comparing phlebotomy by tail tip amputation, facial vein puncture, and tail vein incision in C57BL/6 mice by using physiologic and behavioral metrics of pain and distress.</a> J Am Assoc Lab Anim Sci. 56 (3), 307-317 (2017).</li>
<li>Gong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+and+correlation+of+serum+cortisol+and+corticosterone+under+different+physiological+or+stressful+conditions+in+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Dynamics and correlation of serum cortisol and corticosterone under different physiological or stressful conditions in mice.</a> PLoS One. 10 (2), e0117503(2015).</li>
</ol>

The authors declare no competing interests.

This optimized facial vein blood collection protocol provides an efficient and simple alternative to other commonly used methods, such as tail vein or retro-orbital bleeding. While the protocol is simple and straightforward, a few critical steps must be carefully followed. The most important step to execute properly is the correct placement of the puncture site4. Targeting the appropriate location on the cheek ensures sufficient blood flow with minimal distress to the mouse. Immediately depositing...

Efficient, reproducible, and minimally invasive blood collection is essential for many murine studies, particularly those requiring repeated sampling. This protocol provides a streamlined method for collecting small volumes of blood using facial vein venipuncture, followed by red blood cell lysis, antibody staining, and flow cytometric analysis. This workflow is well-suited for high-throughput applications while minimizing technical complexities.
Many blood collection methods, like retro-orbital bleeding or tail vein incision, can require procedures involving heat,&#160;anesthesia1, and prolonged handling times, which can cause significant distress to the animal2. In contrast, facial vein venipuncture with a lancet offers a simple and rapid alternative that supports ethical and reproducible experimental design. This method has been validated in prior studies as a humane alternative for small-volume blood sampling in mice3.
We illustrate this method by phenotyping immune cells from transgenic and reporter mice, including TdTomato reporter mice and MD4-IgHEL mice. The latter requires staining with antibodies against CD19 and IgMa, an IgM allotype.
While this protocol is developed for blood cellular profiling, it can be easily adapted for other uses, including serum cytokine analysis, specific antibody detection, or glucose measurements, by omitting the red blood cell lysis step. Overall, this technique enhances data quality and experimental efficiency while causing minimal animal distress, making it broadly useful across diverse research applications.

<table><tbody><tr><td>APC anti-mouse CD19 Antibody</td><td>Biolegend</td><td>152410</td><td>CD19 APC</td></tr><tr><td>Centrifuge 5810 R, without rotor, 120 V, 50/60 Hz</td><td>Eppendorf</td><td>22625501</td><td>Centrifuge</td></tr><tr><td>Dermacea Gauze sponges 8 ply 2&quot; x 2&quot;</td><td>Covidien</td><td>441211</td><td>Guaze Ponges</td></tr><tr><td>Disposable Graduated Transfer Pipettes</td><td>Fisherbrand</td><td>13-711-91am</td><td>Transfer pipet</td></tr><tr><td>ErgoOne Single Channel Pipette (200 &amp;micro;L)</td><td>USA Scientific</td><td>7100-2200</td><td>Pipette</td></tr><tr><td>Ethylenediaminetetraacetic Acid (0.5 M Solution/pH 8.0)</td><td>Fisherbrand</td><td>BP2482-100</td><td>EDTA 5 M&amp;nbsp;&amp;nbsp;</td></tr><tr><td>FACSymphony A3 Cell Analyzer</td><td>BD Biosciences</td><td>660937</td><td>Flow Cytometer</td></tr><tr><td>Fisherbrand&amp;nbsp;Analog Vortex Mixer</td><td>Fisherbrand</td><td>02-215-414</td><td>Vortex</td></tr><tr><td>Goldenrod Animal Lancet 3 mm Sterile 5 trays of 200</td><td>Goldenrod</td><td>GR 3MM</td><td>Lancet 3 mm</td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11051-10</td><td>Forceps</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution, 1X without calcium and magnesium</td><td>Corning</td><td>21-021-CVR</td><td>HBSS</td></tr><tr><td>PerCP/Cyanine5.5 anti-mouse IgMa&amp;nbsp;Antibody</td><td>Biolegend</td><td>408612</td><td>IgMa PerCP cy-5.5</td></tr><tr><td>RBC Lysis Buffer (10x)</td><td>Biolegend</td><td>420301</td><td>RBC Lysis Buffer</td></tr><tr><td>Round-Bottom Polystyrene Test Tubes Without Cap</td><td>Fisherbrand</td><td>FB1496110</td><td>Culture tubes</td></tr><tr><td>Sharpie Permanent Markers, Chisel Tip</td><td>Sharpie</td><td>38254PP</td><td>Sharpies&amp;nbsp;</td></tr><tr><td>Tested for USP Sterile Water&amp;nbsp;</td><td>Corning</td><td>25-055-CI</td><td>Cell Culture Grade Water</td></tr><tr><td>TipOne Pipette Tip Refills (200 &amp;micro;L Natural Beveled)</td><td>USA Scientific</td><td>1111-1730</td><td>Pipette tips</td></tr></tbody></table>

facial vein venipuncture for murine blood collection

Facial Vein Venipuncture for Murine Blood Collection

This paper presents an optimized protocol for murine blood collection via facial vein venipuncture using a lancet, offering a rapid, minimally invasive, and cost-effective alternative to other common techniques such as tail vein and retro-orbital sampling. This technique is particularly advantageous for studies requiring repeated sampling, such as mouse phenotyping, serum cytokine analysis, and diabetes research. The protocol is efficient, requires minimal specialized equipment, and is well-suited for high-throughput cellular analysis in mice. Our primary application is the phenotyping of transgenic, congenic, and reporter mice using flow cytometry, demonstrated here with antibody staining and tdTomato reporter mice. The paper outlines the complete procedure, from animal preparation to sample analysis. Key steps include blood collection with a 3 mm lancet, cell lysis, staining, and flow cytometric profiling. Facial vein venipuncture enhances collection efficiency while supporting ethical research practices. This practical and adaptable method is suitable for a wide range of experimental protocols involving murine blood sampling and cellular analysis.

Using flow cytometry, Figure 2 shows lysed blood from wild-type (WT) and PF4-Cre TdTomato reporter mice, prepared using the protocol described above. The first set of flow plots displays side scatter (SSC), which reflects cell granularity, versus forward scatter (FSC), which indicates cell size. Cellular debris and platelets are observed near the origin of the axes, while lymphocytes appear smaller compared to the more granular neutrophils. A distinct separat...

Here, we present a protocol for an optimized blood collection technique in murine subjects through facial vein venipuncture using a lancet. We use this technique alongside flow cytometric assessment to characterize cellular phenotypes of transgenic, congenic, and reporter strains. It is also suitable for serum cytokine analysis and diabetes studies.

This paper presents an optimized protocol for murine blood collection via facial vein venipuncture using a lancet, offering a rapid, minimally invasive, ...

The goal of this research is to present an optimized blood collection method for murine subjects. This method reduces the time and skill needed for small volume blood collection in mice, but still prioritizes ethical and minimally invasive practices. Blood collection methods are often over-complicated.We wanted to simplify it all and show that you can achieve clear lysis with minimal time, effort, and materials. The main advantages of this protocol over other techniques is giving researchers their time back. This method is very quick and requires very few materials or specialized skills to execute.To begin, label each cage card and mouse with a phenotype identifier that matches the corresponding labels on the collection tubes. Mark the tails of mice with a permanent marker to correspond with the markings on the tubes. Open all lancets and gauze sponges required for the procedure, and add one to two milliliters of lysis buffer to each labeled tube.Now using the dominant hand, grasp the mouse by the tail and place it on a wire feeding rack or a flat surface. With the non-dominant index finger and thumb, scruff the mouse by maneuvering from the tail toward the cervical region. Then using the remaining fingers, secure the tail to minimize mouse movement.Next, use the dominant hand to retrieve an open three millimeter lancet. Locate the small hairless spot just beyond the mouth and trace a straight line toward the ear. Stop where the line intersects with the outer corner of the eye.Now, insert the lancet at this intersection to ensure an ample blood supply and use the flat lateral edge of the lancet to collect a single drop of blood. Then deposit the entire lancet with the collected blood into the labeled tube containing red blood cell lysis buffer, and gently agitate the tube to mix the blood completely with the buffer. Using a gauze sponge, apply gentle pressure to the puncture site for 5 to 10 seconds and return the mouse to its cage.Add approximately two milliliters of Hanks'balanced salt solution with 0.5 millimolar EDTA to each sample tube. Place the tubes into the centrifuge and spin at 400 G for five minutes. During this time, prepare the antibody cocktails.After centrifugation, discard the supernatant from each tube. Then using forceps, remove the lancets from the tubes and add 30 microliters of antibody cocktail into each sample. Vortex the samples for two to three seconds to resuspend the cells and incubate the samples at four degrees Celsius for 25 to 30 minutes.Next, turn on the flow cytometer and configure the flow plots according to the experimental requirements. Using a transfer pipette, add 150 to 250 microliters of Hanks'balanced salt solution or other media to each sample. If required, add DAPI at a final concentration of one microgram per milliliter by using 100 microliters of DAPI containing media.Vortex each sample for two to three seconds just before acquisition. Finally, check the reagents and the run settings of the flow cytometer for data acquisition. The flow cytometry of wild-type mice showed no tdTomato signal in platelets, whereas Pf4-Cre reporter mice showed distinct tdTomato positive platelet populations and larger tdTomato positive platelet bound cells.Side scatter versus forward scatter plots from both wild-type and Pf4-Cre mice showed clear separation between cellular debris, lymphocytes, and granulocytes, indicating effective red blood cell lysis and preserved sample structure. CD19-positive IGMA-positive B cells were detected in MD4Ighel mice, whereas wild-type mice lacked the IGMA signal despite CD19 positivity.

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Learn facial vein venipuncture for quick, ethical murine blood collection using a lancet. This simplified method reduces time and skill requirements while enabling serum cytokine analysis and diabetes research.

Facial Vein Venipuncture for Murine Blood Collection

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