2.4 Sample Collection and Processing

2.4 Sample Collection and Processing

2.4.1 Blood Analysis

Twenty and forty days after drug administration, 20 μl of blood is collected from the tail vein of each mouse (fasting for 12 hours before blood collection, but allowed to drink water) and mixed with 20 μl of anticoagulant in a test tube, then sent to the laboratory for routine blood tests.

2.4.2 Bone Marrow Nucleated Cell (BMNC) Extraction

Referencing Tang Peixian’s method [25].

2.4.2.1 Preparation of 0.4% Trypan Blue Solution

0.4 g of trypan blue is added to 100 ml of double-distilled water.

2.4.2.2 Procedure

Forty days after gavage, the mice are euthanized by cervical dislocation. After soaking in 75% alcohol for 3 minutes, the mice are placed on sterile absorbent paper with the abdomen facing up. A small incision is made in the skin at the lower end of the femur, the skin is peeled back to fully expose the femur and tibia (both femurs are taken), the tibia is clamped with forceps, and the muscles along the femur are carefully separated with ophthalmic scissors. At the knee joint, the femur and tibia are cut open, the femur is lifted with forceps, and at the greater trochanter joint, the femur is cut off and placed in a petri dish. Further removal of muscle tissue on the femur is performed, and the left femur is held with rat tooth forceps to remove the bone. A 20-gauge needle is used to puncture the end of the bone, and a 2 ml syringe is used to rapidly inject RPMI-1640 culture medium into the bone marrow cavity to flush out the bone marrow cells, which are collected in a sterile centrifuge tube. The bone marrow cell suspension is repeatedly pushed and pulled with the syringe several times to ensure thorough dispersion of the bone marrow cells, forming a suspension. The bone marrow cell suspension is centrifuged at 1000 r/min to remove the supernatant and fat layer, then re-suspended in RPMI-1640 culture medium at a 1:1 ratio, slowly spread into a centrifuge tube pre-filled with an equal volume of Ficoll-Hypaque lymphocyte separation solution, centrifuged at 3000 r/min for 20 minutes, the middle cloudy single-nucleated cell layer is aspirated and suspended in 20 ml of RPMI-1640 culture medium, centrifuged at 3000 r/min for 10 minutes, the supernatant is discarded, and the precipitated cells are mixed thoroughly with an appropriate amount of RPMI-1640 culture medium to form a cell suspension. 0.5 ml of this suspension is added to a test tube, along with 0.5 ml of 0.4% trypan blue solution, stained for 2–3 minutes, a small amount of the suspension is smeared on a slide, covered with a coverslip, and several random fields are examined under the microscope to count dead and live cells. Dead cells are stained blue by trypan blue [26–28], appearing dark blue under the microscope, while live cells remain colorless and transparent. Only when trypan blue assessment shows cell viability above 95% can nucleated cell counting be performed.

Research on Pei Zhengxue’s Series of Formulas

2.4.3 Preparation of Femoral Pathological Sections