by dextran sedimentation followed by Ficoll density gradient centrifugation

Planning of TLBZT The herbs Cyclopamine 11-deoxojervine found in TLBZT method will be the roots of Actinidia chinensis 30 g, Solanum nigrum 15 g, Duchesnea indica 15 g, Atractylodes macro cephala Koidz 9 g, Poria cocos 15 g, Coix seed 30 g, Mistletoe 15 g, and Scutellaria barbata 30 g. Dozens of herbs were from the herb store in Longhua Hospital based on the original proportion, and decocted twice with 8 fold amount of distilled water for 1 hour. The de coction were obtained, filtered, joined and concen trated to 1. 5 g/mL, and stored at 4 C. For Gas chromatography--mass spectrometry analysis, TLBZT were more extracted with dichloromethane and diethyl ether, and passed through 0. 22 um filter. GC/MS research of TLBZT extract was done by GCMS6800 designed with a DB 5ms column. Helium was used as carrier gas at a consistent flow-rate of just one mL/min. An injection amount of 1 uL was used in splitless mode. Injector and ion source were maintained at 280 C and 230 C, respectively. The mass scan range was 50--500. The GC/MS account of TLBZT is shown in Additional file 1. Figure S1. Cell culture and animal model Gene expression Murine colon carcinoma CT26 cells were obtained from obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences. CT26 cells were grown in DMEM medium with 10 % FBS, penicillin and streptomycin and maintained at 37 C with 52-card CO2 in a humidified atmosphere. Female BALB/c mice were acclimated for starters week and were fed with water ad libitum and animal chow in SPF animal laboratory of Longhua Hospital. The mice were injected s. c. with 1 106 CT26 cells in 100 ul PBS in the right flank. The rats were randomly split into 4 groups, and SL-01 intragastric administered with TLBZT or same level of distilled water, or i, If the tumors were palpable. p. administered with 5 FU, or treated with both 5 Fu and TLBZT. Tumefaction size and length were measured every 3 days by calipers. The cyst volume was determined based on the formula. Tv0. 52 T W2. After three days of treatment, the rats were sacrificed, and the tumors were re moved, considered and put through further studies. All studies involving rats were accepted by the Longhua Hospital Animal Care and Use Committee. TUNEL assay Apoptotic cells were identified by TUNEL assay following a manufacturers guide. Images were captured from the Olympus microscope at 200 magnifica tion. The apoptotic cells were counted by Image Pro Plus 6. 0 pc software. Caspases activities assay The activities of Caspases were detected by Caspase 3, 8 and 9 Activity Assay Kit. Based on the producers protocol, the tumefaction samples were homogenized, and the supernatant were collected and identified protein con centration. Then, the supernatant were respectively incu bated with Ac DEVD pNA, Ac LEHD pNA and Ac IETD pNA in analysis buf fer at 37 C for 2 hours. Eventually, the creation of p nitroaniline was monitored by microplate reader at wave-length of 405 nm.