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Xenograft Tumor Growth Assay
异种移植肿瘤增生分析   

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Abstract

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, Xenograft tumor assay was used to determine the tumorigenic activity of hepatoma cells with ISX over expression on nude mice in vivo.

Materials and Reagents

  1. HCC cells (Hep G2: ATCC® HB-8065 TM and Hep 3B: ATCC® HB-8064 TM)
  2. CAnN.Cg-Foxn1nu/CrlBltw Nude mice
  3. ISX fused with GFP or ISX shRNAi expression constructs
  4. PBS (MDBio)
  5. 2.5% Trypsin (10x) (Gibco , catalog number: 15090-046 )
  6. MEM Culture medium (Gibco, catalog number: 61100-061 )

Equipment

  1. 100 mm2 plate
  2. 15 ml conical tubes
  3. Centrifuge (Beckman Coulter)
  4. Eppendorf
  5. 1 ml syringes (0.45 x 13 mm)
  6. Tissue culture hood
  7. Caliper (Digital Caliper)

Procedure

  1. HCC cells were transfected with ISX fused with GFP or ISX shRNAi expression constructs and selected into stable clones.
  2. Determining the cells number for injection. 5 x 107 cells per ml will be required to trypsinizing (usually a 100% confluent plate of 100 mm2 will yield at least 6 injections at 5 x 106 cells/injection).
  3. Trypsinizing the cells with 1x trypsin solution from needed number of plates to be counted all at once.
  4. Collecting the detached cells in 15 ml conical tubes and spin for 3 min at 300 x g.
  5. To remove the supernatant and re-suspend the cells with 3 ml of culture medium for counting.
  6. To remove three 10 μl aliquots into 3 separate eppendorfs and dilute each 10 μl 1:100 by adding 990 μl of culture medium, mix well for counting.
  7. Then, to remove 10 μl of 1:10 dilutions for counting, counting each of three dilutions and average the three numbers.
  8. Determining the concentration of cells in cells/ml by using the following formula: Average counts x 10,000 x dilution factor (1,000) = #cells/ml.
  9. Determining the volume required to add to achieve final concentration of cells for injection per volume to be injected (i.e. 5 x 106 cells/ml injections).
  10. Spin down 15 ml conical for 3 min at 300 x g.
  11. Discard supernatant and re-suspend the pellet in the previously determined volume from step 8.
  12. Draw up each injection/mouse in 1 ml syringes in the tissue culture hood prior to going to the animal facility. Place the separate syringes each containing 200 μl on ice.
  13. The tumor volume was estimated according to the formula: volume (cm3) = 1/2(L x W2), where L and W are the length and width of the tumor, respectively.

Acknowledgments

This protocol was adapted from Hsu et al. (2013).

References

  1. Hsu, S. H., Wang, L. T., Lee, K. T., Chen, Y. L., Liu, K. Y., Suen, J. L., Chai, C. Y. and Wang, S. N. (2013). Proinflammatory homeobox gene, ISX, regulates tumor growth and survival in hepatocellular carcinoma. Cancer Res 73(2): 508-518.

简介

慢性炎症驱动肝细胞癌(HCC)的引发,但是连接炎症和肿瘤形成的基础机制仍然是模糊的。 在该研究中,使用软琼脂贴壁非依赖性测定来测定具有ISX的肝癌细胞的肿瘤转化活性超过表达或体外敲低。...

材料和试剂

  1. 将HCC细胞(Hep G2:ATCC HB-8065 TM和Hep 3B:ATCC HB-8064 TM sup/)
  2. 裸鼠
  3. ISX与GFP或ISX shRNAi表达构建体融合
  4. PBS(MDBio)
  5. 2.5%胰蛋白酶(10x)(Gibco,目录号:15090-046)
  6. MEM培养基(Gibco,目录号:61100-061)

设备

  1. 100 mm 2
  2. 15 ml锥形管
  3. 离心机(Beckman Coulter)
  4. Eppendorf
  5. 1ml注射器(0.45×13mm)
  6. 组织培养罩
  7. 卡尺(数字卡尺)

程序

  1. 用与GFP或ISX shRNAi表达构建体融合的ISX转染HCC细胞,并选择稳定的克隆
  2. 确定注射的细胞数。 胰蛋白酶化需要5×10 7个细胞/ml(通常100mm 2的100%铺满的平板将在5×10 5个细胞/cm 2下产生至少6次注射, 6 细胞/注射)
  3. 胰蛋白酶消化细胞用1×胰蛋白酶溶液从所需数量的板一次计数
  4. 在15ml锥形管中收集分离的细胞并在300×g下旋转3分钟。
  5. 为了除去上清液并用3ml培养基重新悬浮细胞用于计数
  6. 为了将3个10μl等分试样移入3个独立的eppendorfs中,并通过加入990μl的培养基稀释每个10μl1:100,混匀以便计数。
  7. 然后,为了去除10μl的1:10稀释物用于计数,计数三个稀释度中的每一个并平均三个数字
  8. 使用下式确定细胞的浓度/ml:平均计数×10,000×稀释因子(1,000)=#细胞/ml。
  9. 确定加入以获得待注射的每体积注射的细胞的最终浓度所需的体积(即5×10 6个细胞/ml注射)。
  10. 在300×g下旋转15ml圆锥形3分钟。
  11. 弃去上清液并将沉淀物重悬在步骤8中预先确定的体积中。
  12. 在组织培养罩中,在进入动物设施之前,在1ml注射器中绘制每次注射/小鼠。 将每个含有200μl的单独的注射器置于冰上
  13. 根据以下公式估计肿瘤体积:体积(cm 3)= 1/2(L×W <2),其中L和W是长度和宽度 肿瘤。

致谢

该协议改编自Hsu等人(2013)。

参考文献

  1. Hsu,S.H.,Wang,L.T.,Lee,K.T.,Chen,Y.L.Liu,K.Y.,Suen,J.L.,Chai,C.Y.and Wang,S.N。(2013)。 促炎同源盒基因,ISX,调节肝细胞癌中的肿瘤生长和存活。 Cancer Res 73(2):508-518。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, L. and Hsu, S. (2013). Xenograft Tumor Growth Assay. Bio-protocol 3(20): e948. DOI: 10.21769/BioProtoc.948.
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