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

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

细胞生物学 > 细胞分离和培养 > 细胞生长
作者: Lin Fang
Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USA
For correspondence: cheerfulfang@hotmail.com
Bio-protocol author page: a20
Vol 2, Iss 6, 3/20/2012, 11802 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.130

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

[Abstract] 本方法介绍了通过脂质体(Lipofectamine)建立稳定Caco-2细胞系的方法。

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.

材料与方法

 

1.         脂质体TM转染试剂。(Invitrogen cat# 18324-020)

2.         DMEM培养基,Dulbeccos Modification of Eagles Medium (Invitrogen)

3.         胰蛋白酶(Trypsin-EDTA (GIBCO, Invitrogen)

4.         Opti-MEM (GIBCO, Invitrogen)

5.         DMEM  (GIBCO, Invitrogen)

6.         2 × FBS (GIBCO, Invitrogen)

7.         G418 (Invitrogen))

8.         胰蛋白酶(GIBCO, Invitrogen)

9.         培养皿(VWRI)

10.     Cloning , 真空润滑油, 钳子 (全部灭菌)

 

设备

 

1.         组织培养罩

2.         组织培养孵化器

 

实验步骤

 

1.         10 cm培养皿提前48-72h准备Caco-2细胞,使细胞可以50-70%融合后转染。

例如,如果在30%融合后播撒在10 cm培养皿,细胞会在48小时后融合60%,如果在15%融合后播撒在10 cm培养皿,细胞会在72小时后融合60

注意:

1)       与其他肿瘤细胞株不同,Caco-2细胞接触培养皿非常缓慢,因此总是让细胞生长至少48h达到针对性的融合。

2)       如果使用小于10 cm培养皿,需要测生长速度,由于Caco-2细胞的生长速度不同缓慢程度取决于培养皿大小。

2.          按如下操作转染细胞:

1)        准备混合AB(见下表),之后 混合在一起, 室温放置45 min

2)        C加入AB 混合物

3)        在板(plate)中用D更换生长培养基,滴加方法加入ABC混合物,轻轻摇动板。37°C, 5% CO2孵育5 h

4)        在培养皿中加入E,额外生长数小时。 (全部转染时间是48 h)

 

A

B

C

D

E

DNA (pmol)

(eg. pGL4.17-靶基因)

Opti-MEM

脂质体

Opti-MEM

Opti-MEM

DMEM

w/o FBS

DMEM

w/ 2× 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后,将细胞按1, 1:10, 1:100 以及 1:1000稀释分离在有选择性培养基的10cm培养皿。在的pGL4.17情况下,使用1 mg/ml G418。所选抗生素请参阅供应商项目单。

4.        3周后,单克隆应肉眼可见。其间在培养基颜色变化时更换培养基。一旦细胞克隆可见,你可以根据目的选择挑取单克隆,或是汇集克隆。单克隆操作过程请参阅以下步骤:

1)        PBS清洗板之后吸出。.

2)         用钳子拿起克隆环并将其简要地浸在真空润滑脂中,使油脂在环的边缘而非中心。将克隆环置于克隆的中心并确保环均匀粘在培养皿上。重复此步在所有选取的单克隆上,一般每个构建载体取12个克隆。但避免让培养皿干燥。

3)        每个添加50-100μl胰蛋白酶,于37°C, 5% CO2孵育5min

4)        向环上添加DMEM FBS,吸管来回吸打,并转移细胞到含有选择性培养基的24孔培养皿中。

5)        让细胞生长近一周时间以增长到90%融合。检查细胞间的融合,以免细胞过度拥挤。

6)        一旦细胞在24孔板中达到80 ~ 90%的融合,转移细胞至含有选择性培养基的6cm培养皿中。同时,根据实验设置保留一些细胞供表达测试用。

7)        一旦细胞在6cm培养皿中相互融合,用液氮库存。

 

参考文献

 

1.         Invitrogen Lipefectamine Manual

 

English
中文翻译

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How to cite this protocol: Fang, L. (2012). Stable Caco-2 Cell Line Construction. Bio-protocol 2(6): e130. DOI: 10.21769/BioProtoc.130; Full Text



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7/19/2012 7:00:41 AM  

dear Autor, I can't see what is under E in te table. Thanks

7/25/2012 9:37:36 PM  

Lin Fang (Author)
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.

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