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[Bio101] Lentivirus Infection
[Bio101] 慢病毒感染方法

分子生物学 > DNA > 转染
作者: Nabila Aboulaich
2/20/2011, 18759 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.37

[Abstract]

[Abstract]

Materials and Reagents

 

1.        6 cm tissue culture plates (Fiosher).

2.        Human or mouse cell line and appropriate growth media Reagents required for cell-based assay.

3.        Polybrene (Hexadimethrine bromide; Sigma H 9268 )

4.        Appropriated antibiotics for selection purpose.

 

Equipment

 

1.        Tissue Culture Incubator

 

Procedure

 

Note: Lentiviral infections should be optimized for each cell line and cell-based assay. For example, the following parameters should be tested before starting large-scale infections to determine the optimal conditions for a given experiment:

1)       Cell seeding density.

2)       Amount of lentivirus.

3)       Puromycin concentration.

4)       Timecourse.

1.        Seed cells at appropriate density in 6 mL in 6 cm plates.

1)       Adherent cells: seed 1 day prior to infection.

2)       Suspension cells: seed day of infection in media containing polybrene.

2.        Add virus to cells:

1)         (Adherent cells): Remove growth media and add fresh media containing polybrene. Alternatively, remove a portion of the growth media and supplement with media containing polybrene. Adjust volumes and polybrene concentration to achieve the correct final polybrene concentration (8ug/ml).

3.        Viral infection:

1)       Incubate cells overnight.

2)       Change media 24 hours post-infection. Remove media and replace with 6 mL fresh growth media. If antibiotics selection is desired, use fresh growth media containing antibiotics. Note: Puromycin concentration should be optimized     for each cell line; typical concentrations range from 2-5 μg/mL.

4.        Incubate cells, replacing growth media (with antibiotics, if desired) as needed every few days. Incubation periods are highly dependent on the post-infection assay.

Note: All lentiviral procedures should be carried out in accordance with biosafety requirements of the host institution.

 

 

 

材料和试剂

 

1.      class ="Apple-converted-space">  lang ="EN-US"> 6 cm组织培养板( Fiosher )。

2.      class ="Apple-converted-space">  人或小鼠细胞系和适当的生长培养基基于细胞的测定所需的试剂。

3.      class ="Apple-converted-space"> Polybrene 溴化物; Sigma H 9268) Hexadimethrine >

4.      class ="Apple-converted-space">  lang ="EN-US">用于选择目的的抗生素。

 

设备

 

1.      class ="Apple-converted-space">  lang ="EN-US">组织培养物培养箱

 

过程

 

注意: Lentiviral   感染。例如,在开始大规模感染之前应测试以下参数,以确定给定实验的最佳条件:

1)       "Apple-converted-space"> 细胞接种密度。

2)       "Apple-converted-space"> 的金额<​​/span>。

3)       "Apple-converted-space"> Puromycin  

4)       "Apple-converted-space"> 时间表

1.      class ="Apple-converted-space">  lang ="EN-US"> 6 mL   6厘米 /span>。

1)       "苹果转换 - 空格"> 贴壁细胞:感染前1天的种子。

2)       "Apple-converted-space"> 悬浮细胞:含有 polybrene 的培养基中的感染种子日。

2.      class ="Apple-converted-space">  lang ="EN-US">向单元格添加病毒:

1)        polybrene 的新鲜培养基。 span>。或者,删除一部分生长培养基并补充含有  polybrene 的培养基。调整音量和 polybrene span class ="Apple-converted-space"> polybrene >  浓度(8ug/ml)。

3.      class ="Apple-converted-space">  lang ="EN-US">病毒感染:

1)       "Apple-converted-space"> 孵育细胞过夜。

2)       "Apple-converted-space"> 感染后24小时更改媒体。移除媒体并替换为  mL ; 新鲜生长培养基。如果需要选择抗生素,请使用含有抗生素的新鲜生长培养基。注意: Puromycin   浓度应优化为每个细胞系的       μg/mL

4.      class ="Apple-converted-space">  孵育细胞,根据需要每隔几天更换生长培养基(如果需要,用抗生素)。孵育期高度依赖于感染后测定。

注意:  全部  lentiviral 生物安全 ="Apple转换空间">  要求。

 

 

 

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How to cite this protocol: Aboulaich, N. (2011). Lentivirus Infection. Bio-protocol Bio101: e37. DOI: 10.21769/BioProtoc.37; Full Text



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