A minimally invasive, low-stress method for serial blood collection in aging mice

Abstract

Hematologic analysis is an efficient and valuable tool for real-time health monitoring and immune analysis in mouse aging studies. However, many frequently used blood sampling techniques in mice are incompatible with continuous monitoring with increasing age, as they may involve anesthesia, cause severe stress, or require a high volume of blood. This technical report describes a convenient relatively noninvasive procedure for counting white blood cells in C57Bl/6 mice by an optimized tail blood collection method followed by Wright-Giemsa and Türk staining. This technique can be performed on unanesthetized mice in less than 1 min with minimal stress using only a few microliters of blood. White blood cell analysis can include but is not limited to total and differential white blood cell count and cell morphology. The low blood volume collected is ideal for aging mice in longer-term experiments requiring regular and continuous monitoring.

Keywords: Blood collection protocol; aging mice; hematology; white blood cell differentiation.

Figure 1.

Blood is collected from the tail vein of the mouse, at a blood collection site located down three-fourths of the tail from the base. Approximately 4 µl blood is collected into a heparinized capillary tube. Two-microliter blood is mixed with Turk’s solution for total WBC count. The rest 2 µl blood is used to create blood smear for WBC differentiation. Blue circles: target tail veins, located on both the left and right side of the tail; Red circle: tail artery.

Figure 2.

White blood cell differentiation is performed on blood smear with Wright-Geimsa staining. (a) Lymphocyte is characterized by dark purple circular nucleus; (b) Monocyte is characterized by a purple kidney-shaped nucleus with lysosomes in cytoplasm; (c) Neutrophil is characterized by purple multi-lobular nucleus.

Figure 3.

Serial blood collection and WBC counts in 28-month-old C57BL/6 mice given one dose of cyclophosphamide (100 mg/kg IP) provided significant observations. (a) Total WBC; (b) Lymphocytes; and (c) Neutrophils. The control groups showed no significant variation, while there was a significant decrease in the cyclophosphamide groups. N = 6–8 mice per cohort,*p < 0.05 by student t-test.