Measurement of tumor volume and apoptosis in mouse solid tumor xenograft model

The cell suspension was cultured in 1640 medium containing 10% BSA and 10% penicillin and streptomycin. Mouse lymphoma (EL4) cells (1×106) were then subcutaneously injected into the right hind limb of each animal. Mice bearing tumors of similar sizes were randomly divided into four groups (n=6/group). Mice in the SYKT group received SYKT (1.2 ml/kg/d on days 1, 3 and 5, p.o.). Mice in the DOX group received DOX (3 mg/kg/d on days 2, 4 and 6, i.p.). Mice in the SYKT/DOX group received both SYKT and DOX as aforementioned. Control mice were gavaged with normal saline at an equal volume to SYKT solution on day 1, 3, and 5; and normal saline at an equal volume to DOX solution on day 2, 4 and 6 via peritoneal perfusion. The radii of the developing tumors were measured using Vernier calipers on days 3, 5, 7, 10 and 15, and the tumor volume was calculated using the formula V=4/3πr₁2r₂, where r₁ and r₂ denote the radii of the tumor in two different planes. For the tumor cell apoptosis assay, the animals were sacrificed and xenografts were isolated on days 7, 10 and 15. The tumors were homogenated after thoroughly rinsing with ice-cold PBS. The tumor stroma was then fully degraded with collagenase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in an incubator for 1.5 h. After the tumor tissue was dispersed with ice-cold PBS containing 1% BSA, a single-cell suspension was acquired via filtration through a 400 mm filter. The cells were stained with Fluorescein-12-dUTP and propidium iodide (PI)-PE for TUNEL apoptosis assay and analyzed using flow cytometry. Procedures for preparing the sample ready for flow cytometry detection were briefly described as follows: Formaldehyde-fixed cells (15 min, 4°C) were centrifuged at 755 × g for 5 min, followed by rinsing twice with PBS. The supernatant was then discarded and 70% pre-cold ethanol was added to fix the cells for 15 min at 4°C, followed by centrifugation at 755 × g for 5 min, and a rinse twice with PBS. Double distilled water (30 µl), bio-dNTP (1 µl), TdT (terminal deoxynucleotidyl transferase) (5 µl) and TdT buffer solution (10 µl) were added in proper sequence, mixed and cultured at 37°C for 30 min. Then, 100 µl of FITC-avidin was added and the mixture was kept away from light, at room temperature for 30 min, followed by centrifugation at 1500 rpm at 4°C for 5 min. The product was washed twice with PBS containing 0.1% tritonX-100 and then 50 µl of PI was added. The mixture was kept away from light, at room temperature for 20 min prior to testing with flow cytometry to evaluate the apoptotic rate. All samples were analyzed in triplicate using FlowCytomixPro Software (Version 2.1).