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Chemosensitivity Assay
化疗敏感性测试   

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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

  1. Osteosarcom cell line MNNG/HOS# from the cell bank of the Chinese Academy of Sciences (Shanghai, China)
  2. Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DF) (Sigma-Aldrich, catalog number: D8900 )
  3. Fetal Bovine Serum (FBS) (Hyclone, catalog number: GUC SH30084 )
  4. NaCl (Wako Chemicals USA, catalog number: 191-01665 )
  5. KCl (Wako Chemicals USA, catalog number: 163-03545 )
  6. Na2HPO4 (Wako Chemicals USA, catalog number: 197-02865 )
  7. KH2PO4 (Wako Chemicals USA, catalog number: 169-04245 )
  8. Trypsin (0.05%) (Sigma-Aldrich, catalog number: T4799 )
  9. EDTA (0.04%) (Sigma-Aldrich, catalog number: E6758 )
  10. Trypsin inhibitor (0.05%) (Sigma-Aldrich, catalog number: T9128 )
  11. CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, catalog number: G3582 )
  12. Cisplatin (Sigma-Aldrich, catalog number: P4394 )
  13. Adriamycin (Sigma-Aldrich, catalog number: D1515 )
  14. Agarose (Sigma-Aldrich, catalog number: A9539 )
  15. Type I collagen (Life Technologies, Gibco®, catalog number: 17100-017 )
  16. Collagenase I (Sigma-Aldrich, catalog number: C3867 )
  17. Collagenase IV (Life Technologies, Gibco®, catalog number: 17104-019 )
  18. Insulin (Sigma-Aldrich, catalog number: I5500 )
  19. Apo-Transferrin (Sigma-Aldrich, catalog number: T2252 )
  20. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  21. Ethanolamine (Sigma-Aldrich, catalog number: E0135 )
  22. Sodium selenite (Sigma-Aldrich, catalog number: S5261 )
  23. BSA (Sigma-Aldrich, catalog number: A6003 )
  24. Oleic acid (Sigma-Aldrich, catalog number: O1383 )
  25. Heparin (Sigma-Aldrich, catalog number: H3149 )
  26. L-Ascorbic acid 2-phosphate trisodium salt (Wako Chemicals USA, catalog number: 323-44822 )
  27. Milli Q water
  28. TGF-β1 signaling inhibitor SB431542 (Biovision, catalog number: 1674-1 )
  29. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D5879 )
  30. PBS without Ca2+ and Mg2+ (see Recipes)
  31. Serum-Free medium (see Recipes)
  32. Cancer stem cell medium (see Recipes)

Equipment

  1. Centrifuge (Thermo Heraeus Megafuge 1.0)
  2. CO2 cell culture incubator (NuAire, model: nu8700E )
  3. Microscope (Nikon corporation Japan, model: Nikon Eclipse Ti-U )
  4. Microplate Reader 680 (Bio-Rad Laboratories, model: 168-1001 )
  5. Coulter Counter (Beckman Coulter, model: Z1 )
  6. Volume adjustable micropipet (Gilson 1-10 μl, model: FA10002M ; 10-100 μl, model: FA10004M ; 100-1,000 μl, model: FM10006M )
  7. Multichannel pipettor (BIOHIT mline pipettor, model: 725140 )
  8. 10 ml Glass pipette (BrandTech, model: 27713 )
  9. Electric pipette (BrandTech, model: accu-jet pro 2026330 )
  10. 96-well culture plate (Greiner Bio-One GmbH, catalog number: 655180 )
  11. 24-well plate (Greiner Bio-One GmbH, catalog number: 665180 )
  12. 50 ml centrifuge tube

Procedure

  1. For serum cultured monolayer cells in 96-well plate
    1. Maintain stock culture of monolayer cells in DF medium supplemented with 10% FBS (Figure 1).


      Figure 1. Osteosarcoma cells MNNG/HOS#

    2. When monolayer cells are 80~90% confluent, discard medium and wash cells once with 2 ml 1x PBS, and then digest cells with 1 ml trypsin-EDTA for 2-5 min at 37 °C.
    3. Check cell status under microscope. When the cells are disassociated, terminate digestive reaction with the same volume of FBS.
    4. Pipette cells into 50 ml centrifuge tube, and collect cells by centrifugation at 800 rpm for 5 min.
    5. Suspend cell pellet by DF medium without antibiotics. DF medium used for chemosensitivity assay are antibiotics free.
    6. Count the cells by Coulter Counter.
    7. Adjust cell density at 50,000 cells/ml by DF medium supplemented with 5% FBS.
    8. Dispense 100 μl of cell suspension (50,000 cells/ml) in a 96-well plate.
    9. Incubate the cells for 24 h in a humidified incubator (at 37 °C, 5% CO2).
    10. Add 1 μl of various concentrations of adriamycin or cisplatin into each well of the 96-well assay plate containing the cells, and shake the 96-well plate horizontally to mix the drugs (do not need to change the medium).
    11. Incubate the cells for 72 h in a humidified incubator (at 37 °C, 5% CO2).
    12. Thaw the CellTiter 96® AQueous One solution reagent. It should take approximately 90 minutes at room temperature, or 10 min in a water bath at 37 °C, to completely thaw the 20 ml size.
    13. Pipette 20 μl of CellTiter 96® AQueous One solution reagent into each well of the 96-well assay plate containing the cells in 100 μl of culture medium, and shake the 96-well plate horizontally to mix the reagent (Do not need to change the medium).
    14. Incubate the plate at 37 °C for 2 h in a humidified incubator (at 37 °C, 5% CO2).
    15. Record the absorbance at 490 nm using a Microplate Reader 680.

  2. For serum-free cultured monolayer cells in 24-well plate
    1. Preparation for 24-well plate:
      1. Prepare 1% type I collagen solution with chilled 1x PBS.
      2. Add 0.5 ml collagen solution into each well of 24-well plate.
      3. Stand 24-well plate for 1 h at room temperature.
      4. Aspirate excess collagen solution, and rinse the well with 1x PBS once.
      5. Aspirate 1x PBS completely.
    2. Maintain stock culture of monolayer cells in DF medium supplemented with 10% FBS.
    3. When monolayer cells are 80~90% confluent, discard medium and wash cells once in 2 ml 1x PBS.
    4. Digest cells with 1 ml trypsin-EDTA for 2-5 min at 37 °C.
    5. Check cell status under microscope. When the cells are disassociated, terminate digestive reaction with the same volume of trypsin inhibitor.
    6. Pipette cells into 50 ml centrifuge tube, and collect cell by centrifugation at 800 rpm for 5 min.
    7. Suspend cell pellet by DF medium without antibiotics. DF medium used for chemoresistance assay are antibiotics free.
    8. Count the cells by Coulter Counter.
    9. Adjust cell density at 20,000/ml by serum free DF medium.
    10. Add 1 ml cell solution onto each well of 24-well plate.
    11. Incubate the cell for 24 h.
    12. Add various concentrations of adriamycin or cisplatin into each well of 24-well plate containing the cells, and shake the 24-well plate horizontally to mix the drugs (do not need to change the medium).
    13. Incubate cells for another 3 days.
    14. Discard supernatant, rinse each well by 1x PBS.
    15. Add 0.5 ml trypsin-EDTA to each well.
    16. Collect disassociated cells, and count cell number by Coulter Counter.
  3. For spheroid cells
    1. Preparation for 96-well plate:
      1. Dissolve agarose powder in Milli Q water.
      2. Prepare 0.7% agarose solution, sterilized by autoclave for use.
      3. Dispense 20 μl of 0.7% agarose into each well of 96-well plate.
      4. Stand 96-well plate for 20 min at room temperature.
      5. The 96-well plate could be used after agarose solidification.
    2. Maintain stock culture of osteosarcoma spheroid cells in cancer stem cell medium (Figure 2).


      Figure 2. Osteosarcoma spheres. The spheroid cells are suspended cultured in cancer stem cell medium.

    3. When spheres reach 100 μm in diameter, pipette spheres into 50 ml centrifuge tube and collect cells by centrifugation at 500 rpm for 3 min.
    4. Discard the supernatant; digest spheres with the mixture of trypsin (0.05%), collagenase I (0.1%) and collagenase IV (0.1%) for 2 min at 37 °C.
    5. When spheres are digested to be single cells, terminate digestive reaction with 0.05% trypsin inhibitor.
    6. Collect cells by centrifugation at 800 rpm for 5 min. Suspend cell pellet by cancer stem cell medium without antibiotics. Cancer stem cell medium used for chemosensitivity assay are antibiotics free.
    7. Count the cells by Coulter Counter.
    8. Adjust cell density at 50,000 cells/ml by cancer stem cell medium.
    9. Dispense 100 μl of cell suspension (50,000 cells/ml) onto each agarose coated well of 96-well plate.
    10. Incubate the cells for 24 h in a humidified incubator (at 37 °C, 5% CO2).
    11. Add 1 μl of various concentrations of adriamycin or cisplatin into each well of 96-well plate containing the cells, and shake the 96-well plate horizontally to mix the drugs (do not need to change the medium).
    12. Incubate the cells for 72 h in a humidified incubator (at 37 °C, 5% CO2).
    13. Thaw the CellTiter 96® AQueous One solution reagent.
    14. Pipette 20 μl of CellTiter 96® AQueous One solution reagent into each well of the 96-well plate containing the cells in 100 μl of culture medium, and shake the 96-well plate horizontally to mix the reagent (Do not need to change the medium).
    15. Incubate the plate at 37 °C for 2 h in a humidified incubator (at 37 °C, 5% CO2).
    16. Record the absorbance at 490 nm using a Microplate Reader 680.
  4. For spheroid cells which are pre-treated with TGF-β1 signaling inhibitor SB431542.
    1. Preparation for 96-well plate:
      1. Dissolve agarose powder in Milli Q water.
      2. Prepare 0.7% agarose solution, sterilized by autoclave for use.
      3. Dispense 20 μl of 0.7% agarose into each well of 96-well plate.
      4. Stand 96-well plate for 20 min at room temperature.
      5. The 96-well plate could be used after agarose solidification.
    2. Maintain stock culture of osteosarcoma spheroid cells in cancer stem cell medium.
    3. Osteosarcoma spheroid cells are pre-treated with 376 nM SB431542 (dissolved in 0.01% DMSO) for 3 days (or pre-treated with 0.01% of DMSO for control) in cancer stem cell medium.
    4. Pipette spheres into 50 ml centriguge tube and collect cells by centrifugation at 500 rpm for 3 minutes.
    5. Discard the supernatant; digest spheres with the mixture of trypsin (0.05%), collagenase I (0.1%) and collagenase IV (0.1%) for 2 min at 37 °C.
    6. When spheres are digested to be single cells, terminate digestive reaction with 0.05% trypsin inhibitor.
    7. Collect cells by centrifugation at 800 rpm for 5 min. Suspend cell pellet by cancer stem cell medium without antibiotics. Cancer stem cell medium used for chemoresistance assay are antibiotics free.
    8. Determine cell number by Coulter Counter.
    9. Adjust cell density at 50,000 cells/ml by cancer stem cell medium.
    10. Dispense 100 μl of cell suspension (5,000 cells/well) onto each agarose coated well of 96-well plate.
    11. Incubate the cells for 24 h in a humidified incubator (at 37 °C, 5% CO2).
    12. Add 1 μl of various concentrations of adriamycin or cisplatin into each well of 96-well plate containing the cells, and shake the 96-well plate horizontally to mix the drugs (do not need to change the medium).
    13. Incubate the cells for 72 h in a humidified incubator (at 37 °C, 5% CO2).
    14. Thaw the CellTiter 96® AQueous One solution reagent.
    15. Pipette 20 μl of CellTiter 96® AQueous One solution reagent into each well of the 96-well plate containing the cells in 100 μl of culture medium, and shake the 96-well plate horizontally to mix the reagent (Do not need to change the medium).
    16. Incubate the plate at 37 °C for 2 h in a humidified incubator (at 37 °C, 5% CO2).
    17. Record the absorbance at 490 nm using a Microplate Reader 680.

Recipes

  1. PBS without Ca2+ and Mg2+
    8 g/L NaCl
    0.2 g/L KCl
    1.42 g/L Na2HPO4
    0.27 g/L KH2PO4
  2. Serum-Free medium
    2.5 mg/L Insulin
    5 mg/L apo-Transferrin
    1 mM 2-mercaptoethanol
    1 mM Ethanolamine
    1 μM Sodium selenite
  3. Cancer stem cell medium
    Insulin (10 mg/L)
    Apo-Transferrin (5 mg/L)
    2-mercaptoethanol (1 mM)
    Ethanolamine (1 mM)
    Sodium selenite (2 μM)
    BSA (9.4 mg/L)
    Oleic acid (1.88 mg/L)
    Heparin (150 μg/L)
    L-Ascorbic acid 2-phosphate trisodium salt (10 mg/L)

Acknowledgments

This study was supported in part by a grant from the program of the State High-Tech Development Project (No. 2008AA092604), National Basic Research Program (No. 2009CB945400) and Guangdong Planning Project of Science and Technology (No. 2009B030803037).

References

  1. Adhikari, A. S., Agarwal, N., Wood, B. M., Porretta, C., Ruiz, B., Pochampally, R. R. and Iwakuma, T. (2010). CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance. Cancer Res 70(11): 4602-4612.
  2. Ginestier, C., Liu, S., Diebel, M. E., Korkaya, H., Luo, M., Brown, M., Wicinski, J., Cabaud, O., Charafe-Jauffret, E., Birnbaum, D., Guan, J. L., Dontu, G. and Wicha, M. S. (2010). CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J Clin Invest 120(2): 485-497. 
  3. Haraguchi, N., Ishii, H., Mimori, K., Tanaka, F., Ohkuma, M., Kim, H. M., Akita, H., Takiuchi, D., Hatano, H., Nagano, H., Barnard, G. F., Doki, Y. and Mori, M. (2010). CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 120(9): 3326-3339. 
  4. Zhang, H., Wu, H., Zheng, J., Yu, P., Xu, L., Jiang, P., Gao, J., Wang, H. and Zhang, Y. (2013). Transforming growth factor beta1 signal is crucial for dedifferentiation of cancer cells to cancer stem cells in osteosarcoma. Stem Cells 31(3): 433-446.

简介

化学抗性是癌症干细胞的特殊性质之一,其是化疗失败的主要原因,并且在各种癌症(包括骨肉瘤)的复发中起重要作用。 最广泛使用的用于评价化学抗性的测定法是修饰的细胞增殖测定法。 将等量的细胞接种到96孔培养板或24孔板上,然后将不同浓度的抗癌药物加入每个孔中。 之后,测量细胞密度或细胞活性,观察抗癌药物对癌细胞或癌症干细胞的作用。 细胞的化学抗性更强,细胞密度或细胞活性更高。 在这里,我们描述了骨肉瘤细胞和骨肉瘤干样细胞的化学敏感性测定。

关键字:肿瘤干细胞, 化疗敏感性, 无血清培养, 球, MTT比色法

材料和试剂

  1. 骨髓细胞细胞系MNNG/HOS#来自中国科学院细胞库(中国上海)
  2. Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham(DF)(Sigma-Aldrich,目录号:D8900)
  3. 胎牛血清(FBS)(Hyclone,目录号:GUC SH30084)
  4. NaCl(Wako Chemicals USA,目录号:191-01665)
  5. KCl(Wako Chemicals USA,目录号:163-03545)
  6. Na 2 HPO 4(Wako Chemicals USA,目录号:197-02865)

  7. (Wako Chemicals USA,目录号:169-04245)。
  8. 胰蛋白酶(0.05%)(Sigma-Aldrich,目录号:T4799)
  9. EDTA(0.04%)(Sigma-Aldrich,目录号:E6758)
  10. 胰蛋白酶抑制剂(0.05%)(Sigma-Aldrich,目录号:T9128)
  11. CellTiter 96 AQueous One Solution Cell Proliferation Assay kit(Promega Corporation,目录号:G3582)
  12. 顺铂(Sigma-Aldrich,目录号:P4394)
  13. 阿霉素(Sigma-Aldrich,目录号:D1515)
  14. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  15. I型胶原(Life Technologies,Gibco ,目录号:17100-017)
  16. 胶原酶I(Sigma-Aldrich,目录号:C3867)
  17. 胶原酶IV(Life Technologies,Gibco ,目录号:17104-019)
  18. 胰岛素(Sigma-Aldrich,目录号:I5500)
  19. Apo-Transferrin(Sigma-Aldrich,目录号:T2252)
  20. 2-巯基乙醇(Sigma-Aldrich,目录号:M7522)
  21. 乙醇胺(Sigma-Aldrich,目录号:E0135)
  22. 亚硒酸钠(Sigma-Aldrich,目录号:S5261)
  23. BSA(Sigma-Aldrich,目录号:A6003)
  24. 油酸(Sigma-Aldrich,目录号:O1383)
  25. 肝素(Sigma-Aldrich,目录号:H3149)
  26. L-抗坏血酸2-磷酸三钠盐(Wako Chemicals USA,目录号:323-44822)
  27. Milli Q水
  28. TGF-β1信号传导抑制剂SB431542(Biovision,目录号:1674-1)
  29. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D5879)
  30. PBS无Ca 2 + 和Mg 2 + (参见配方)
  31. 无血清培养基(见配方)
  32. 癌干细胞培养基(见配方)

设备

  1. 离心机(Thermo Heraeus Megafuge 1.0)
  2. CO 2细胞培养箱(NuAire,型号:nu8700E)
  3. 显微镜(Nikon corporation Japan,型号:Nikon Eclipse Ti-U)
  4. 微量板阅读器680(Bio-Rad Laboratories,型号:168-1001)
  5. Coulter计数器(Beckman Coulter,型号:Z1)
  6. 体积可调微量移液管(Gilson1-10μl,型号:FA10002M;10-100μl,型号:FA10004M;100-1,000μl,型号:FM10006M)
  7. 多通道移液器(BIOHIT mline pipettor,型号:725140)
  8. 10ml玻璃移液管(BrandTech,型号:27713)
  9. 电动移液器(BrandTech,型号:accu-jet pro 2026330)
  10. 96孔培养板(Greiner Bio-One GmbH,目录号:655180)
  11. 24孔板(Greiner Bio-One GmbH,目录号:665180)
  12. 50ml离心管

程序

  1. 对于96孔板中血清培养的单层细胞
    1. 在补充有10%FBS的DF培养基中保持单层细胞的原种培养物(图1)
    2. 用补充有5%FBS的DF培养基调节细胞密度为50,000细胞/ml
    3. 在96孔板中分配100μl细胞悬浮液(50,000个细胞/ml)。
    4. 孵育细胞在潮湿的孵化器(37°C,5%CO 2)24小时。
    5. 向含有细胞的96孔测定板的每个孔中加入1μl不同浓度的阿霉素或顺铂,并水平摇动96孔板以混合药物(不需要更换培养基)。
    6. 在潮湿的培养箱(37°C,5%CO 2)中孵育细胞72小时。
    7. 解冻CellTiter 96 ® AQueous One溶液试剂。在室温下需要约90分钟,或在37℃的水浴中10分钟,以完全解冻20毫升大小。
    8. 吸取20μl的CellTiter 96 AQueous One溶液试剂到96孔测定板的每个孔中,其含有在100μl培养基中的细胞,并水平摇动96孔板以混合试剂(不需要更换介质)。
    9. 在37℃在潮湿的培养箱(37℃,5%CO 2)中孵育平板2小时。
    10. 使用Microplate Reader 680记录490nm处的吸光度
  2. 对于24孔板中的无血清培养的单层细胞
    1. 24孔板的制备:
      1. 用冷却的1×PBS制备1%I型胶原溶液。
      2. 向24孔板的每个孔中加入0.5ml胶原溶液
      3. 将24孔板在室温下放置1小时。
      4. 吸出过量的胶原蛋白溶液,并用1×PBS清洗一次。
      5. 完全吸出1×PBS
    2. 在补充有10%FBS的DF培养基中维持单层细胞的原种培养物
    3. 当单层细胞为80〜90%汇合时,弃去培养基并在2ml 1×PBS中洗涤细胞一次
    4. 用1ml胰蛋白酶-EDTA在37℃消化细胞2-5分钟。
    5. 在显微镜下检查细胞状态。当细胞分离时,用相同体积的胰蛋白酶抑制剂终止消化反应
    6. 吸取细胞到50ml离心管,并收集细胞通过在800 rpm离心5分钟。
    7. 通过没有抗生素的DF培养基悬浮细胞沉淀。用于化学抗性测定的DF培养基是不含抗生素的。
    8. 通过Coulter计数器计数细胞。
    9. 用无血清DF培养基调节细胞密度为20,000/ml
    10. 向24孔板的每个孔中加入1ml细胞溶液
    11. 孵育细胞24小时。
    12. 向含有细胞的24孔板的每个孔中加入各种浓度的阿霉素或顺铂,并水平摇动24孔板以混合药物(不需要更换培养基)。
    13. 将细胞再培养3天。
    14. 弃去上清液,用1x PBS冲洗每个孔
    15. 向每个孔中加入0.5ml胰蛋白酶-EDTA
    16. 收集取消关联的单元格,并通过Coulter计数器计数单元格数量。
  3. 对于球体细胞
    1. 96孔板的制备:
      1. 将琼脂糖粉末溶解在Milli Q水中
      2. 准备0.7%琼脂糖溶液,通过高压灭菌消毒使用。
      3. 将20μl的0.7%琼脂糖分配到96孔板的每个孔中。
      4. 在室温下放置96孔板20分钟。
      5. 96孔板可以在琼脂糖凝固后使用。
    2. 在癌干细胞培养基中维持骨肉瘤球体细胞的储备培养物(图2)

      图2.骨肉瘤球。球状细胞悬浮在癌干细胞培养基中培养。

    3. 当球体直径达到100μm时,将移液管球体装入50ml离心管中,并通过在500rpm离心3分钟收集细胞。
    4. 弃去上清液;消化球与胰蛋白酶(0.05%),胶原酶I(0.1%)和胶原酶IV(0.1%)的混合物在37℃下2分钟。
    5. 当球体消化为单细胞时,用0.05%胰蛋白酶抑制剂终止消化反应
    6. 通过在800rpm离心5分钟收集细胞。通过没有抗生素的癌症干细胞培养基悬浮细胞沉淀。用于化学敏感性测定的癌干细胞培养基是不含抗生素的。
    7. 通过Coulter计数器计数细胞。
    8. 用癌干细胞培养基调节细胞密度为50,000个细胞/ml
    9. 分配100微升细胞悬液(50,000细胞/毫升)到96孔板的每个琼脂糖涂覆的孔。
    10. 孵育细胞在潮湿的孵化器(37°C,5%CO 2)24小时。
    11. 向含有细胞的96孔板的每个孔中加入1μl不同浓度的阿霉素或顺铂,并水平摇动96孔板以混合药物(不需要更换培养基)。
    12. 在潮湿的培养箱(37°C,5%CO 2)中孵育细胞72小时。
    13. 解冻CellTiter 96 ® AQueous One溶液试剂。
    14. 吸取20μl的CellTiter 96 AQueous One溶液试剂到含有100μl培养基中的细胞的96孔板的每个孔中,并水平摇动96孔板以混合试剂(不需要更换介质)。
    15. 在37℃在潮湿的培养箱(37℃,5%CO 2)中孵育平板2小时。
    16. 使用Microplate Reader 680记录490nm处的吸光度
  4. 对于用TGF-β1信号传导抑制剂SB431542预处理的球状体细胞。
    1. 96孔板的制备:
      1. 将琼脂糖粉末溶解在Milli Q水中
      2. 准备0.7%琼脂糖溶液,通过高压灭菌消毒使用。
      3. 将20μl的0.7%琼脂糖分配到96孔板的每个孔中。
      4. 在室温下放置96孔板20分钟。
      5. 96孔板可以在琼脂糖凝固后使用。
    2. 保持肿瘤干细胞培养基中骨肉瘤球体细胞的储备培养。
    3. 用癌症干细胞培养基中的376nM SB431542(溶解于0.01%DMSO中)预处理骨肉瘤球体细胞3天(或用0.01%DMSO预处理用于对照)。
    4. 移液球到50ml离心管中,并通过在500rpm离心3分钟收集细胞
    5. 弃去上清液;消化球与胰蛋白酶(0.05%),胶原酶I(0.1%)和胶原酶IV(0.1%)的混合物在37℃下2分钟。
    6. 当球体消化为单细胞时,用0.05%胰蛋白酶抑制剂终止消化反应
    7. 通过在800rpm离心5分钟收集细胞。通过没有抗生素的癌症干细胞培养基悬浮细胞沉淀。用于化学抗性测定的癌干细胞培养基是不含抗生素的。
    8. 通过Coulter计数器确定细胞数量。
    9. 用癌干细胞培养基调节细胞密度为50,000个细胞/ml
    10. 分配100微升细胞悬液(5,000个细胞/孔)到96孔板的每个琼脂糖涂覆的孔。
    11. 孵育细胞在潮湿的孵化器(37°C,5%CO 2)24小时。
    12. 向含有细胞的96孔板的每个孔中加入1μl不同浓度的阿霉素或顺铂,并水平摇动96孔板以混合药物(不需要更换培养基)。
    13. 在潮湿的培养箱(37°C,5%CO 2)中孵育细胞72小时。
    14. 解冻CellTiter 96 ® AQueous One溶液试剂。
    15. 吸取20μlCellTiter 96 AQueous One溶液试剂到含有100μl培养基中的细胞的96孔板的每个孔中,并水平摇动96孔板以混合试剂(不需要更换介质)。
    16. 在37℃在潮湿的培养箱(37℃,5%CO 2)中孵育平板2小时。
    17. 使用Microplate Reader 680记录490nm处的吸光度

      食谱

      1. PBS,不含Ca 2+ 和Mg 2 +
        8g/L NaCl
        0.2 g/L KCl
        1.42g/L Na 2 HPO 4
        0.27g/L KH 2 PO 4 sub/
      2. 无血清培养基
        2.5 mg/L胰岛素 5mg/L载脂蛋白转运蛋白
        1mM 2-巯基乙醇 1mM乙醇胺 1μM亚硒酸钠
      3. 癌干细胞培养基
        胰岛素(10mg/L)
        载脂转铁蛋白(5mg/L) 2-巯基乙醇(1mM) 乙醇胺(1mM)
        亚硒酸钠(2μM)
        BSA(9.4mg/L)
        油酸(1.88mg/L)
        肝素(150μg/L)
        L-抗坏血酸2-磷酸三钠盐(10mg/L)

      致谢

      本研究部分得到了国家高新技术开发项目(No. 2008AA092604),国家基础研究计划(No. 2009CB945400)和广东科技规划项目(No. 2009B030803037)资助。

      参考文献

      1. Adhikari,A.S.,Agarwal,N.,Wood,B.M.,Porretta,C.,Ruiz,B.,Pochampally,R.R。和Iwakuma,T。(2010)。 CD117和Stro-1可识别与转移和耐药性相关的骨肉瘤肿瘤起始细胞。 Cancer Res 70(11):4602-4612。
      2. Ginestier,C.,Liu,S.,Diebel,ME,Korkaya,H.,Luo,M.,Brown,M.,Wicinski,J.,Cabaud,O.,Charafe-Jauffret,E.,Birnbaum, ,Guan,JL,Dontu,G。和Wicha,MS(2010)。 CXCR1封锁可有选择性地靶向体外人类乳腺癌干细胞和异种移植物。 J Clin Invest 120(2):485-497。 
      3. Haraguchi,N.,Ishii,H.,Mimori,K.,Tanaka,F.,Ohkuma,M.,Kim,HM,Akita,H.,Takiuchi,D.,Hatano,H.,Nagano, ,GF,Doki,Y。和Mori,M。(2010)。 CD13是人类肝癌干细胞的治疗靶点 J Clin Inves t 120(9):3326-3339。 
      4. Zhang,H.,Wu,H.,Zheng,J.,Yu,P.,Xu,L.,Jiang,P.,Gao,J.,Wang,H.and Zhang, 转化生长因子β1信号对于癌细胞对骨肉瘤中癌干细胞的去分化至关重要。 a> Stem Cells 31(3):433-446。
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引用:Zhang, H. and Zhang, Y. (2013). Chemosensitivity Assay. Bio-protocol 3(17): e891. DOI: 10.21769/BioProtoc.891.
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