TDS - BD Biosciences

Collecting Blood Samples
Page 5 of 8
(Version 030626)
MicroFlow BASIC Kit (Mouse)
Method: Collect mouse peripheral blood using a method approved by the Institutional Animal Care and Use
Committee (IACUC). The tail vein is a common site for obtaining peripheral blood from a rat. The subsequent
fixation procedure and micronucleus analysis is also effective for blood obtained from the orbital sinus or by cardiac
puncture.
Volume: Collect only 60–120 µl of blood (whether taking blood from the tail vein, orbital sinus, or heart) from each
animal. It is important to obtain no more than 120 µl of blood since too much blood may compromise the flow
cytometric analysis. It is also important that no debris be introduced into the sample.
Anticoagulant: Use only Solution B as the anticoagulant; do not substitute a different anticoagulant. Collect drops of
blood from the tail vein directly into tubes of Solution B. If a capillary tube or syringe is used to draw blood, pre-wet it
with Solution B. Have extra tubes containing Solution B ready in case of a spill. Warning: Some capillary tubes
include a clot activator that will cause aggregation and make the samples unanalyzable.
1. Tubes containing 0.35 ml Solution B (anticoagulant) should be stored at 4°C until use. Approximately one hour
before collecting blood, move these tubes to room temperature and maintain at room temperature throughout
the blood collection procedure.
2. For tail vein bleeding, place a mouse or group of mice (up to 5 animals) under a heat lamp (100 watt bulb
placed 6 to 8 inches above the animals) for 5–10 minutes to dilate the tail veins.
3. Shake the microcentrifuge tube immediately before bleeding each animal to coat the inside of the tube with
Solution B.
4. Make an incision in the tail vein using a sterile blade.
5. Place the corresponding labeled tube of Solution B under the incision, and collect approximately three drops of
blood (~120 µl). [Again, too much blood can be as great of a problem as not having enough. The volume of
Solution B is appropriate for approximately three drops of blood. Varying from this amount may lead to samples
which cannot be analyzed.]
To prevent clotting, hold the tube so that the blood drops directly into Solution B and does not contact the side
of the tube. It is important to make the incision such that the blood is free flowing, and falls directly into
Solution B in a minimal amount of time (< 10 seconds). Apply moderate pressure to the wound with a laboratory
tissue to stop the bleeding.
6. Cap the tube and invert the tube several times to mix the blood with Solution B. The blood/Solution B mixture
can be stored at room temperature for up to 6 hours before fixing.
7. Repeat steps 3–6 for the remaining mice.
Fixing Conditions
It is extremely important that the tubes containing Solution A and fixed blood remain ultracold (–75 to –85 °C) and
do not come in contact with vapors from dry ice. CO2 vapor causes carbonation and cellular aggregation. For this
same reason, fixative should not be stored in a freezer containing dry ice, and fixation should not occur on dry ice.
To avoid this problem, Solution A should be taken directly from the freezer.
If you are unable to fix blood samples DIRECTLY from the –75 to –85 °C freezer as described in the Fixing
Procedure, it is imperative that you do not fix the samples off dry ice under any circumstances without first calling
Litron at (585) 442-0930 or sending an email to info@litronlabs.com. Litron will provide you with a detailed
alternative fixing protocol. Without this alternative protocol, blood samples CANNOT be fixed appropriately and will
be compromised, and flow cytometric analysis CANNOT be performed. Litron Laboratories is not responsible for
samples prepared using a procedure other than the one described in this manual or supplied by Litron as an
alternative.