Facial Vein Venipuncture for Murine Blood Collection

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.