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Karyotype Analysis
染色体组型分析   

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Abstract

A chromosome is the structure that organizes DNA and protein in cells. It is a single piece of coiled DNA containing coding and non-coding sequences. Human cells have 23 pairs of chromosomes including 22 pairs of autosomes and one pair of sex chromosome, giving a total of 46 per cell. In tumor cells, chromosomal instability has been considered to be one of the hallmarks of tumor formation. Here we use the karyotype analysis to separate the chromosomes and observe the chromosomes in tumor cells with a microscope.

Keywords: Chromosome(染色体), Chromosomal instability(染色体不稳定性), Tumor formation(肿瘤的形成)

Materials and Reagents

  1. Cell lines: FaDu (ATCC, catalog number: HTB-43TM )
  2. Trypsine (Life Technologies, Gibco®, catalog number: 15400-054 )
  3. 10% fetal bovine serum (Thermo Fisher Scientific, catalog number: SH30071.03 )
  4. 1% Penicillin-Streptomycin (10,000 U/ml; 10,000 μg/ml) (Life Technologies, Gibco®, catalog number: 15140-122 )
  5. Colcemid (10 μg/ml) (Life Technologies, Gibco®, catalog number: 15212-012 )
  6. Hypotonic solution (0.075 M KCl) (J.T.Baker®, catalog number: 7447-40-7 )
  7. Glacial acetic acid (J.T.Baker®, catalog number: 64-19-7 )
  8. Fixative (see Recipes)
  9. Growth medium (see Recipes)

Equipment

  1. 10 cm cell culture dishes (Trueline Valve Corpotation, catalog number: TR4002 )
  2. Centrifuge tube
  3. Microscope (OLYMPUS, model: DP71 )
  4. CO2 incubator
  5. Centrifuge at room temperature (using “relative centrifugal force, rcf”)
  6. Water bath

Procedure

Day 1

  1. Seed 5 x 105 cells on 10 cm cell culture dishes for attachment.
  2. Cells were incubated at 37 °C, 5% CO2 for 48 h.

Day 3

  1. Add 0.1 ml colcemid, which can collapse mitotic spindles and prevent the completion of mitosis, to each dish and mix gently. Incubated at 37 °C, 5 % CO2 for 2 h.
  2. Transfer medium to centrifuge tubes from the cell culture dishes. Use PBS to wash the dishes, and remove PBS. Then, add 1 ml 0.1% Trypsine into the dishes at 37 °C, 5% CO2 for 2 min. Also, transfer the mixture (Trypsine and cells) into the centrifuge tubes and mix with the medium which is transferred to centrifuge tubes before we use PBS to wash the dishes. Further, centrifuge at 100 rcf for 10 min at room temperature.
  3. Discard the supernatant and leave 0.5 ml medium to mix the pellet gently.
  4. Resuspend the pellet in 5-7 ml 37 °C hypotonic solution and mix thoroughly. Incubate in water bath at 37 °C for 10 min.
  5. Centrifuge at 100 rcf for 10 min at room temperature.
  6. Discard the supernatant and leave 0.5 ml solution to mix the pellet gently.
  7. Resuspend the pellet in 5 ml cold fixative (drop by drop and flick the tube between drops to prevent cell clumping).
  8. Put the centrifuge tube on ice at least 20 min.
  9. Centrifuge at 100 rcf for 10 min at room temperature.
  10. Discard the supernatant and add 3-5 ml cold fixative.
  11. Repeat steps 9-10 three times or until the supernatant is clear and the pellet become white.
  12. After the final centrifugation, suspend the cells in a few drops of cold fixative to give a slightly opaque suspension.
  13. Drop 1-2 drops onto wet and clean slides (Before that, the slides have to be rinsed by 1-2 drops of cold fixative.)
  14. Dry the slides at room temperature.
  15. Observe the chromosomes with the microscope (Olympus DP71 with 200x magnification).
  16. Count the number of chromosomes about 50 cells (A normal human cell have 23 pairs of chromosomes.).
    Note: Chromosomes center in a scope, we define that “one cell”.

Representative data



Figure 1. Representative pictures of karyotype analysis in FaDu cell line (see Chou et al., 2013)

Recipes

  1. Fixative
    Methanol and glacial acetic acid (3:1) to be made fresh and chilled before using
  2. Growth medium
    RPMI 1640 supplemented with:
    10% fetal bovine serum
    1% Penicillin Streptomycin (10,000 U/ml; 10,000 μg/ml)

Acknowledgments

This protocol has been adapted from Chou et al. (2013).

References

  1. Chou, C. H., Yang, N. K., Liu, T. Y., Tai, S. K., Hsu, D. S., Chen, Y. W., Chen, Y. J., Chang, C. C., Tzeng, C. H. and Yang, M. H. (2013). Chromosome instability modulated by BMI1-AURKA signaling drives progression in head and neck cancer. Cancer Res 73(2): 953-966.

简介

染色体是组织细胞中的DNA和蛋白质的结构。 它是含有编码和非编码序列的单片卷曲DNA。 人类细胞具有23对染色体,包括22对常染色体和一对性染色体,每个细胞总共46个染色体。 在肿瘤细胞中,染色体不稳定性已被认为是肿瘤形成的标志之一。 这里我们使用核型分析来分离染色体,并用显微镜观察肿瘤细胞中的染色体

关键字:染色体, 染色体不稳定性, 肿瘤的形成

材料和试剂

  1. 细胞系:FaDu(ATCC,目录号:HTB-43 TM
  2. 胰蛋白酶(Life Technologies,Gibco ,目录号:15400-054)
  3. 10%胎牛血清(Thermo Fisher Scientific,目录号:SH30071.03)
  4. 1%青霉素 - 链霉素(10,000U/ml;10,000μg/ml)(Life Technologies,Gibco ,目录号:15140-122)
  5. 秋水仙碱(10μg/ml)(Life Technologies,Gibco ,目录号:15212-012)
  6. 低渗溶液(0.075M KCl)(J.T.Baker ,目录号:7447-40-7)
  7. 冰醋酸(J.T.Baker ,目录号:64-19-7)
  8. 固定剂(见配方)
  9. 生长培养基(参见食谱)

设备

  1. 10cm细胞培养皿(Trueline Valve Corpotation,目录号:TR4002)
  2. 离心管
  3. 显微镜(OLYMPUS,型号:DP71)
  4. CO <2>孵化器
  5. 在室温下离心(使用"相对离心力,rcf")
  6. 水浴

程序

第1天

  1. 在10cm细胞培养皿上接种5×10 5个细胞用于附着。
  2. 将细胞在37℃,5%CO 2下孵育48小时

第3天

  1. 添加0.1毫升秋水仙素,其可以崩溃有丝分裂纺锤体和防止有丝分裂的完成,每个菜和轻轻混合。 在37℃,5%CO 2孵育2小时
  2. 转移培养基到离心管从细胞培养皿。使用PBS清洗盘子,并删除PBS。然后,在37℃,5%CO 2下,向培养皿中加入1ml 0.1%胰蛋白酶2分钟。此外,将混合物(胰蛋白酶和细胞)转移到离心管中,并与转移到离心管中的培养基混合,之后我们使用PBS洗涤培养皿。此外,在室温下以100rcf离心10分钟
  3. 弃去上清液,留下0.5ml培养基,轻轻混合沉淀
  4. 重悬在5-7毫升37°C低渗溶液中的沉淀和彻底混合。在37℃水浴中孵育10分钟
  5. 在室温下以100rcf离心10分钟。
  6. 弃去上清液并留下0.5ml溶液,轻轻混合沉淀
  7. 将沉淀重悬于5ml冷固定剂中(逐滴并在滴之间轻弹管子以防止细胞聚集)。
  8. 将离心管置于冰上至少20分钟。
  9. 在室温下以100rcf离心10分钟
  10. 弃去上清液并加入3-5ml冷固定剂
  11. 重复步骤9-10三次或直到上清液澄清,颗粒变白
  12. 最后离心后,将细胞悬浮在几滴冷固定剂中,得到稍微不透明的悬浮液
  13. 滴1-2滴湿和干净的幻灯片(之前,幻灯片必须用1-2滴冷固定剂冲洗。)
  14. 在室温下干燥载玻片。
  15. 用显微镜观察染色体(Olympus DP71,放大200倍)。
  16. 计数大约50个细胞的染色体数目(正常人细胞有23对染色体)。
    注意:染色体位于示波器中心,我们定义为"一个单元格"。

代表数据



图1. FaDu细胞系中核型分析的代表性图片 (参见Chou等人,2013)

食谱

  1. 固定剂
    使用
    前,将甲醇和冰醋酸(3:1)制成新鲜的并冷冻
  2. 生长培养基
    RPMI 1640补充:
    10%胎牛血清 1%青霉素链霉素(10,000U/ml;10,000μg/ml)

致谢

该协议已经改编自Chou等人。(2013)。

参考文献

  1. Chou,C.H.,Yang,N.K.,Liu,T.Y.,Tai,S.K.,Hsu,D.S.,Chen,Y.W.,Chen,Y.J.,Chang,C.C.,Tzeng,C.H.and Yang, 由BMI1-AURKA信号调节的染色体不稳定驱动头颈癌的进展 < em> Cancer Res 73(2):953-966。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chou, C. and Yang, M. (2014). Karyotype Analysis. Bio-protocol 4(10): e1129. DOI: 10.21769/BioProtoc.1129.
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zelong li
BGI living bank
10/17/2017 12:02:24 PM Reply