果蝇Schneider 2细胞磷酸钙法转染   

刘爱国刘爱国*刘敏刘敏*朱健朱健  (*contributed equally to this work)
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Original research article

A brief version of this protocol appeared in:
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摘要:果蝇Schneider 2 (S2) 细胞系是从果蝇早期胚胎分离出来的一类血淋巴细胞,也是果蝇仅有的能进行细胞实验的几种细胞系中最常见的一种。因此,掌握S2细胞的转染方法对于果蝇细胞生物学研究非常重要。目前,市面上针对S2细胞的脂质体转染试剂相对较少且价格昂贵,转染效率一般也难以超过20%。磷酸钙转染方法成本低廉,并且有着不逊于脂质体转染试剂的效率,是一种性价比非常高的转染方法。

关键词: 果蝇, S2细胞系, 磷酸钙, 细胞转染

材料与试剂

  1. 6孔细胞培养板 (Thermo Fisher Scientific, Nunc, catalog number: 140675)
  2. 24孔细胞培养板 (Thermo Fisher Scientific, Nunc, catalog number: 142475)
  3. 1.5 ml塑料离心管 (Corning, AXYGEN, catalog number: MCT-150-C)
  4. 果蝇S2细胞
  5. 一次性无菌过滤装置 (Thermo Fisher Scientific, Nelgene, catalog number: 1660045)
  6. CaCl2 (Merck, Sigma-Aldrich, catalog number: 223506)
  7. NaCl (Merck, Sigma-Aldrich, catalog number: S5886)
  8. KCl (Merck, Sigma-Aldrich, catalog number: P9333)
  9. Na2HPO4 (Merck, Sigma-Aldrich, catalog number: S7907)
  10. Glucose (Thermo Fisher Scientific, Gibco, catalog number: 15023021)
  11. HEPES (free acid) (VWR Life Science, AMRESCO, catalog number: 0511)
  12. NaOH (Merck, Sigma-Aldrich, catalog number: 221465)
  13. 0.25 M CaCl2 (见溶液配方)
  14. 2× HEPES (见溶液配方)

仪器设备

  1. 震荡混匀仪 (Vortex) (其林贝尔, model: VORTEX-5)

实验步骤

  1. 待细胞生长密度达到90%左右时,传代至6孔板,每孔约2 ml培养基,至少两小时后,当细胞密度达到40%左右时可进行转染 (图1)。


    图1. S2细胞生长密度达到90% (左) 和40% (右)

  2. 取1.5 ml离心管,将3 μg左右的质粒或RNA稀释在100 μl 0.25 M CaCl2中。
  3. 取1.5 ml离心管加入100 μl 2× HEPES,将CaCl2/DNA混合物逐滴加入,边加边用震荡混匀仪混匀。(也可以边加边用手指弹匀)
  4. 将混合物静置10~20分钟,然后逐滴加入到细胞中,轻轻摇晃细胞培养板将其混匀。
  5. 约12小时后,为细胞更换新鲜的培养基。
  6. 对于24孔板,可使用约35 μl左右的CaCl2和2× HEPES,其他培养皿可酌情增减。

注意事项

  1. 磷酸钙转染需使用含血清的培养基,对于无血清培养基培养的细胞,磷酸钙转染效率极差。
  2. CaCl2和2× HEPES配制后需分装保存,转染时若两者混合后产生沉淀,则说明溶液的pH异常,需重新调节或配制。

溶液配方

  1. 0.25 M CaCl2
    过滤,分装后-20 °C保存
  2. 2× HEPES (500 ml)
    8.00 g NaCl
    0.35 g KCl
    0.20 g Na2HPO4
    1.00 g Glucose
    5.00 g HEPES
    加双蒸水至500 ml,之后用1 ml左右10 N NaOH调节pH至7.10。过滤,分装后-20 °C保存

致谢

本实验室的工作得到细胞增殖与分化重点教育部重点实验室、膜生物学国家重点实验室、生命科学联合中心、国家自然科学基金和科学技术部的资金支持。本实验方案改编自本实验室的发表文章Du等 (2016) 和 Liu等 (2016)。

参考文献

  1. Du, J., Zhang, J., He, T., Li, Y., Su, Y., Tie, F., Liu, M., Harte, P. J. and Zhu, A. J. (2016). Stuxnet facilitates the degradation of polycomb protein during development. Dev Cell 37(6): 507-519.
  2. Liu, M., Li, Y., Liu, A., Li, R., Su, Y., Du, J., Li, C. and Zhu, A. J. (2016). The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development. Elife 5: e17200.
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:刘爱国, 刘敏, 朱健. (2019). 果蝇Schneider 2细胞磷酸钙法转染. Bio-101: e1010257. DOI: 10.21769/BioProtoc.1010257.
How to cite: Liu, A. G., Liu, M. and Zhu, A. J. (2019). Ca-phosphate-mediated Transient Transfection method in Drosophila S2 Cells. Bio-101: e1010257. DOI: 10.21769/BioProtoc.1010257.
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