化疗敏感性测试

Haixia  Zhang; Yan  Zhang

doi:10.21769/BioProtoc.891

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Peer-reviewed

Chemosensitivity Assay

化疗敏感性测试

Haixia Zhang

Yan Zhang email

发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.891 浏览次数: 12022

评审: Fanglian HeLin FangAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Mar 2013

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Abstract

Chemoresistance is one of the special properties of cancer stem cells, which is the main cause of chemotherapy failure and plays an important role in the recurrence of various cancers including osteosarcoma. The most widely used assay for evaluating chemoresistance is the modified cell proliferation assay. An equal number of cells are seeded onto 96-well culture plates or 24-well plates, and then different concentrations of anti-cancer drugs are added into each well. After that, measure the cell density or cell activity to observe the effect of anti-cancer drugs to cancer cells or cancer stem cells. The chemoresistance of cells is stronger, the cell density or cell activity is higher. Here, we describe chemosensitivity assays for osteosarcoma cells and osteosarcoma stem-like cells.

Keywords: Cancer stem cell (肿瘤干细胞)

Chemosensitivity (化疗敏感性)

Serum-free culture (无血清培养)

Sphere (球)

MTT assay (MTT比色法)

Materials and Reagents

Osteosarcom cell line MNNG/HOS# from the cell bank of the Chinese Academy of Sciences (Shanghai, China)
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DF) (Sigma-Aldrich, catalog number: D8900 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: GUC SH30084 )
NaCl (Wako Chemicals USA, catalog number: 191-01665 )
KCl (Wako Chemicals USA, catalog number: 163-03545 )
Na₂HPO₄ (Wako Chemicals USA, catalog number: 197-02865 )
KH₂PO₄(Wako Chemicals USA, catalog number: 169-04245 )
Trypsin (0.05%) (Sigma-Aldrich, catalog number: T4799 )
EDTA (0.04%) (Sigma-Aldrich, catalog number: E6758 )
Trypsin inhibitor (0.05%) (Sigma-Aldrich, catalog number: T9128 )
CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, catalog number: G3582 )
Cisplatin (Sigma-Aldrich, catalog number: P4394 )
Adriamycin (Sigma-Aldrich, catalog number: D1515 )
Agarose (Sigma-Aldrich, catalog number: A9539 )
Type I collagen (Life Technologies, Gibco^®, catalog number: 17100-017 )
Collagenase I (Sigma-Aldrich, catalog number: C3867 )
Collagenase IV (Life Technologies, Gibco^®, catalog number: 17104-019 )
Insulin (Sigma-Aldrich, catalog number: I5500 )
Apo-Transferrin (Sigma-Aldrich, catalog number: T2252 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
Ethanolamine (Sigma-Aldrich, catalog number: E0135 )
Sodium selenite (Sigma-Aldrich, catalog number: S5261 )
BSA (Sigma-Aldrich, catalog number: A6003 )
Oleic acid (Sigma-Aldrich, catalog number: O1383 )
Heparin (Sigma-Aldrich, catalog number: H3149 )
L-Ascorbic acid 2-phosphate trisodium salt (Wako Chemicals USA, catalog number: 323-44822 )
Milli Q water
TGF-β1 signaling inhibitor SB431542 (Biovision, catalog number: 1674-1 )
Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D5879 )
PBS without Ca²⁺ and Mg²⁺ (see Recipes)
Serum-Free medium (see Recipes)
Cancer stem cell medium (see Recipes)

Equipment

Centrifuge (Thermo Heraeus Megafuge 1.0)
CO₂ cell culture incubator (NuAire, model: nu8700E )
Microscope (Nikon corporation Japan, model: Nikon Eclipse Ti-U )
Microplate Reader 680 (Bio-Rad Laboratories, model: 168-1001 )
Coulter Counter (Beckman Coulter, model: Z1 )
Volume adjustable micropipet (Gilson 1-10 μl, model: FA10002M ; 10-100 μl, model: FA10004M ; 100-1,000 μl, model: FM10006M )
Multichannel pipettor (BIOHIT mline pipettor, model: 725140 )
10 ml Glass pipette (BrandTech, model: 27713 )
Electric pipette (BrandTech, model: accu-jet pro 2026330 )
96-well culture plate (Greiner Bio-One GmbH, catalog number: 655180 )
24-well plate (Greiner Bio-One GmbH, catalog number: 665180 )
50 ml centrifuge tube