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Stable Caco-2 Cell Line Construction
稳定Caco-2细胞系的建立   

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Abstract

This protocol describes the construction of Caco-2 stable cell lines using the Lipofectamine transfection method.

Materials and Reagents

  1. Caco-2 cell
  2. Lipofectamine TM transfection reagent. (Life Technologies, Invitrogen™, catalog number: 18324-020 )
  3. Dulbecco’s Modification of Eagles Medium (DMEM) (Life Technologies, Invitrogen™)
  4. Trypsin-EDTA (Life Technologies, Invitrogen™/Gibco®)
  5. Opti-MEM (Life Technologies, Invitrogen™/Gibco®)
  6. 2x FBS (Life Technologies, Invitrogen™/Gibco®)
  7. G418 (Life Technologies, Invitrogen™)
  8. Trypsin (Life Technologies, Invitrogen™/Gibco®)
  9. Cloning ring, vacuum grease (all autoclaved)
  10. Trypsin

Equipment

  1. Tissue culture hood
  2. Culture dish (VWRI)
  3. 10 cm dish
  4. Tissue culture incubator
  5. 24-well culture dishes
  6. Forceps (autoclaved)

Procedure

  1. Prepare Caco-2 in 10 cm dish 48-72 h ahead of time so that cells can be 50-70% confluent upon transfection.
    For example, if seeded at 30% confluent in 10 cm dish, the cells will be 60% confluent 48 h later, if seeded at 15% confluent in 10 cm dish, the cells will be 60% confluent 72 h later.
    Notes:
    1. Different from other cancer cell lines, the Caco-2 cells attach the culture dishes really slowly, so always allow at least 48 h for the cell to grow to targeted confluence.
    2. Please test the growing speed if using dishes smaller than 10 cm as the growing speed of Caco-2 cells is slower in different extent depending on the dish size.

  2. Transfect the cells as following:
    1. Prepare mix A and B (see Table), then mix together, leave at room temperature for 45 min.
    2. Add C to AB mixture.
    3. Replace growth medium with D in the plates, add ABC mixture drop wise while gently swirling the plate. Incubate 37 °C, 5% CO2 for 5 h.
    4. Add E to dish, and grow for additional hours (total transfection time is 48 h)

    A
    B
    C
    D
    E
    DNA (pmol)
    (eg. pGL4.17-target gene)
    Opti-MEM
    Lipofectamine
    Opti-MEM
    Opti-MEM
    DMEM
    w/o FBS
    DMEM
    w/ 2x FBS
    1.125 pmol
    508.5 μl
    40.68 μl
    508.5 μl
    1525.5 μl
    2542.5 μl
    5.1 ml


  3. 48 h later, split the cells into 10 cm dish with selection medium at 1, 1:10, 1:100 and 1:1,000 dilutions of the cells. In the case of pGL4.17, 1 mg/ml G418 was used. Please refer to the provider menu for your selection antibiotic.

  4. Three weeks later, single colonies should be visible to the naked eye. At this time, change medium when medium color changes. Once colonies become visible, you can choose to pick single colonies, or pool the colonies depending on your purpose. Please see following steps for single colony procedure:
    1. Wash the plate with PBS then aspirate.
    2. Pick up a cloning ring with forceps and dip it briefly in vacuum grease so that the grease is in edge of the ring but not in the center. Place the cloning ring so the colony is in the center and make sure the ring sticks evenly well to the dish. Repeat this on all the colonies you intend to pick and normally 12 colonies for each construct. But avoid let the dish dry.
    3. Add to each of them 50-100 μl trypsin and incubate at 37 °C 5% CO2 for 5 min.
    4. Add DMEM with FBS to the ring, pipette up and down and transfer the cells to 24-well culture dishes with selection medium.
    5. Allow approximately one week for the cells to grow to 90% confluent. Check the cell confluence in between to avoid cell being over crowded.
    6. Once the cells in 24-well plate reach 80 ~ 90% confluences transfer the cells to 6 cm dish with selection medium. At the same time, save some cells for expression test depending on your experiment settings.
    7. Once the cells in 6 cm dish reach confluence make liquid nitrogen stock.

Acknowledgments

This protocol was adapted from Reference 1 in the Eric Sibley lab, Department of Pediatrics, Stanford University, CA, USA. Funding from the NIH supported this work.

References

  1. Invitrogen Lipefectamine Manual. http://tools.lifetechnologies.com/content/sfs/manuals/Lipofectamine_Reag_protocol.pdf.

简介

该协议描述了使用Lipofectamine转染方法构建Caco-2稳定细胞系。

材料和试剂

  1. Caco-2单元格
  2. Lipofectamine TM转染试剂。 (Life Technologies,Invitrogen TM,目录号:18324-020)
  3. Dulbecco's Modification of Eagles Medium(DMEM)(Life Technologies,Invitrogen TM)
  4. 胰蛋白酶-EDTA(Life Technologies,Invitrogen TM/Gibco )
  5. Opti-MEM(Life Technologies,Invitrogen TM/Gibco )
  6. 2x FBS(Life Technologies,Invitrogen TM/Gibco )
  7. G418(Life Technologies,Invitrogen TM)
  8. 胰蛋白酶(Life Technologies,Invitrogen TM/Gibco )
  9. 克隆环,真空润滑脂(全部高压灭菌)
  10. 胰蛋白酶

设备

  1. 组织培养罩
  2. 培养皿(VWRI)
  3. 10厘米培养皿
  4. 组织培养孵化器
  5. 24孔培养皿
  6. 钳(高压灭菌)

程序

  1. 提前48-72小时在10厘米培养皿中准备Caco-2,以便转染后细胞可以50-70%汇合。
    例如,如果在10cm培养皿中以30%汇合接种,则细胞将在48小时后60%汇合,如果在10cm培养皿中以15%汇合接种,则细胞将在72小时后60%汇合。
    注意:
    1. 与其他癌细胞系不同,Caco-2细胞非常缓慢地附着培养皿,因此总是允许至少48小时以使细胞生长到目标汇合。
    2. 如果使用小于10厘米的菜肴,请测试生长速度,因为Caco-2细胞的生长速度根据培养皿尺寸的不同而有所不同。

  2. 转染细胞如下:
    1. 制备混合物A和B(见表),然后混合在一起,在室温下放置45分钟
    2. 将C加到AB混合物中。
    3. 在板中用D代替生长培养基,在轻轻旋转板的同时滴加ABC混合物。 在37℃,5%CO 2孵育5小时
    4. 将E加入培养皿中,再培养几小时(总转染时间为48小时)

    A
    B
    C
    D
    E
    DNA(pmol)
    (例如pGL4.17-靶基因)
    Opti-MEM
    脂质体
    Opti-MEM
    Opti-MEM
    DMEM
    w/o FBS
    DMEM
    w/2x FBS
    1.125 pmol
    508.5微升
    40.68μl
    508.5微升
    1525.5微升
    2542.5微升
    5.1 ml


  3. 48小时后,将细胞分裂成10cm培养皿,选择培养基为1:1:10,1:100和1:1,000稀释的细胞。 在pGL4.17的情况下,使用1mg/ml G418。 请参考您选择的抗生素的提供者菜单。

  4. 三个星期后,肉眼可见单个菌落。此时,当介质颜色变化时更换介质。一旦殖民地变得可见,您可以选择挑选单个殖民地,或根据您的目的集合殖民地。请参见单菌落操作的以下步骤:
    1. 用PBS洗板,然后吸出。
    2. 用镊子拿起一个克隆环,将其简单地浸入真空润滑脂中,使润滑脂在环的边缘,但不在中心。放置克隆环,使菌落在中心,并确保环均匀地粘到菜。对您打算选择的所有菌落重复此操作,每个构建体通常有12个菌落。但避免让菜干。
    3. 向其中每个加入50-100μl胰蛋白酶,并在37℃,5%CO 2下孵育5分钟。
    4. 加入DMEM与FBS到环,移液器上下和转移细胞到24孔培养皿与选择培养基。
    5. 允许大约一周使细胞生长至90%汇合。检查两者之间的细胞汇合,以避免细胞过度拥挤。
    6. 一旦24孔板中的细胞达到80〜90%汇合,将细胞转移到具有选择培养基的6cm培养皿中。 同时,根据实验设置保存一些单元格进行表达式测试。
    7. 一旦6cm盘中的细胞达到汇合,就形成液氮储备

致谢

该协议改编自Eric Sibley实验室的参考文献1,美国加利福尼亚州斯坦福大学儿科系。 NIH的资助支持了这项工作。

参考文献

  1. Invitrogen Lipefectamine手册。 http://tools.lifetechnologies.com/content/sfs/manuals/Lipofectamine_Reag_protocol.pdf 。
  • English
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fang, L. (2012). Stable Caco-2 Cell Line Construction. Bio-protocol 2(6): e130. DOI: 10.21769/BioProtoc.130.
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dear Autor, I can't see what is under E in te table. Thanks
7/19/2012 7:00:41 AM Reply
Lin Fang
Department of Pediatrics, School of Medicine, Stanford University, USA

E is 5.1ml DMEM with 2x FBS. (Here 2 x FBS refers to the double amount of whatever working concentration of FBS you use for regular cell culturing.). I hope I answered your question.

7/25/2012 9:37:36 PM