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This method is used for making high titer human immunodeficiency virus type-1 (HIV-1) virus stock for subsequent infection assays. The amplification of T-tropic HIV-1 virus (IIIB strain) uses the CD4+ T cell line H9.

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Amplification of HIV-1 Infectious Virus in BL3 Lab
在BL3实验室扩增HIV-1传染性病毒

微生物学 > 微生物-宿主相互作用 > 病毒
作者: Xin Wang
Xin WangAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: xinwang73@hotmail.com
Bio-protocol author page: a26
Vol 2, Iss 5, 3/5/2012, 6023 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.102

[Abstract] This method is used for making high titer human immunodeficiency virus type-1 (HIV-1) virus stock for subsequent infection assays. The amplification of T-tropic HIV-1 virus (IIIB strain) uses the CD4+ T cell line H9.

[Abstract] 这一方法制得的高浓度1型人类免疫缺陷病毒(HIV-1)可用于后续的传染性实验分析。T-tropic HIV-1病毒 (IIIB菌) 的扩增用的是CD4+ T细胞株系H9。

Materials and Reagents

  1. CD4+ T cell line H9 cells (ATCC HTB-176 )
  2. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH30071.03 HI )
    Note: This particular FBS has been tested by the author, but may be substituted with FBS from different suppliers as desired by users.
  3. Penicillin-Streptomycin liquid (Life Technologies, Gibco®, catalog number: 15070-063 )
  4. Cell culture media: RPMI1640 (Life Technologies, Gibco®, catalog number: 11875-093 ) (see Recipes)

Equipment

  1. Bench-top centrifuges
  2. CO2 incubator
  3. -80 °C freezer
  4. 50 ml conical tubes (BD Biosciences, Falcon®, catalog number: 35-2070 )
  5. 5, 10, 25-ml pipet (BD Biosciences, Falcon®, catalog number: 35-7501 , 35-7554 , 35-7556 )
  6. T-75, T-175 cell culture flask (BD Biosciences, Falcon®, catalog number: 35-3136 , 35-3112 )
  7. CryoTube Vials (NUNC, 377267 )

Procedure

Day 1

  1. Dilute 2 x 106 uninfected H9 cells in 2 ml of culture medium (final concentration 1 x 106 cells/ml) in a 50-ml falcon tube.
  2. Add 1 ml of HIV-1 virus (IIIB strain) to the falcon tube.
  3. Shake gently every 15 min for 1 h.
  4. Pipet this miniculture into 50 ml of culture medium in a T-75 cell culture flask.
  5. Incubate at 37 °C for 4 days.
    Day 5
  6. Add 175 ml fresh media to each of 2 new large falcon T-175 cell culture flasks.
  7. Add 25 ml of 4-day culture to each flask.
  8. Incubate at 37 °C for 3 days.
    Day 8
  9. HIV-1 infected cells (400 ml) were transferred into 10 x 50-ml falcon tubes using a 25-ml pipet (40 ml per tube).
  10. Centrifuge in secondary containers at 1,300 rpm for 8 min.
  11. Carefully remove supernatant using the vacuum line with pipet tip attached to a 10-ml pipet (fill collection flask with bleach).
  12. Add 2 ml of fresh media to each tube and loosen the cell pellets.
  13. Collect all cells in 2 tubes and rinse the other 8 tubes with fresh media and add into those 2 tubes.
  14. Add fresh media to each of these 2 tubes to a final volume of 40 ml.
  15. Centrifuge in secondary containers at 1,300 rpm for 8 min.
  16. Remove supernatant.
  17. Add 10 ml of fresh media to each flask and loosen cell pellet.
  18. Combine into one tube.
  19. Beat up cells for 20 sec using tube rack.
  20. Centrifuge in secondary containers at 2,000 rpm for 8 min.
  21. Transfer supernatant using pipet into a fresh 50-ml falcon tube (Supernatant 1).
  22. Add 10 ml of fresh media to tube with cell pellet and loosen cell pellet.
  23. Beat up cells again using tube rack.
  24. Centrifuge in secondary containers at 2,000 rpm for 8 min.
  25. Transfer supernatant using pipet into a fresh 50-ml falcon tube (Supernatant 2).
  26. Tranfer Supernatant 1 and 2 into small CryoTube vials using a 5-ml pipet (1 ml each tube) (label tubes with IIIB, date and either Sup1 or Sup 2).
  27. Store in -80 °C freezer.

Notes

The experiment using HIV-1 needs to be done in a BSL-3 lab and the personnel require special training for working with HIV-1 virus.

Recipes

  1. Culture medium (500 ml)
    RPMI1640 medium
    500 ml
    FBS
    50 ml
    Penicillin-streptomycin 
    5 ml

Acknowledgments

The author thanks Dutschman, GE and Dr. Yung-Chi Cheng at the Department of Pharmacology, Yale School of Medicine for the adaptation of this method and their support on the research of anti-HIV-1 drug development.

References

  1. Dutschman, G. E., Grill, S. P., Gullen, E. A., Haraguchi, K., Takeda, S., Tanaka, H., Baba, M. and Cheng, Y. C. (2004). Novel 4'-substituted stavudine analog with improved anti-human immunodeficiency virus activity and decreased cytotoxicity. Antimicrob Agents Chemother 48(5): 1640-1646.

材料和试剂

 

1.       抗生素及培养基

1)          细胞培养基:RPMI1640 (Gibco, 11875-093)

2)          牛胎血清 (FBS) (Hyclone, SH30071.03HI

3)          青霉素-链霉素液体 (Gibco, 15070063)

注释:上面提到的FBS可用性已经得到作者的验证,用户也可以根据自己的需要用别的FBS代替。

2.       其它材料:

1)          50 ml 圆锥管(BD Falcon, 35-2070

2)          冷冻管(CryoTube Vials (NUNC, 377267)

3)          T-75, T-175 细胞培养瓶 (BD Falcon, 35-3136, 35-3112)

4)          5, 10, 25-ml 吸量管 (BD Falcon, 35-7501, 35-7554, 35-7556)

 

设备

 

1.       CO2 培养箱

2.       BECKMAN 离心机和转子 (BECKMAN)

3.       -80 °C冰箱

 

步骤 

 

1.       第一天

1)            2 x106未被感染的H9细胞在2 ml培养基(RPMI 1640, 终浓度1 x106 cells/ml)中稀释,装于50ml的圆锥管。

2)            往圆锥管中加入1 ml HIV-1病毒 (IIIB)

3)            每隔15分钟轻摇一次,持续1小时。

4)            用吸量管把小摇的细胞培养液转接到50ml的培养基中,装于T-75细胞培养瓶。

5)            37 °C 培养4天。

2.       第五天

1)            175ml新的培养基于两个新的T-175细胞培养瓶。

2)            往每个培养瓶中加入25ml培养了4天的细菌培养液。

3)            37 °C培养3天。

3.       第八天

1)            25ml的吸量管把HIV-1感染的细胞(400ml)分装到1050ml的圆锥管中(每管40ml)。

2)            置于另一个较大的离心管中,以转速1300 rpm离心8分钟。

3)            10ml的吸量管的尖头小心地吸除上清液(使收集瓶中保留细胞体)。

4)            往每管2ml新鲜的培养基,并轻摇悬起细胞。

5)            把八个管中的细胞都收集到剩下的两个管中,并用新鲜的培养基把管中残留的细胞都洗到两个管中。

6)            往这两个管中加入新鲜的培养基至体积到40ml

7)            置于另一个较大的离心管中,以转速1300 rpm离心8分钟。

8)            倒掉上清液。

9)            往每管10ml新鲜的培养基,并悬起细胞。

10)        两管合为一管。

11)        在圆锥管架上震荡细胞20秒。

12)        置于另一个较大的离心管中,以转速2000 rpm离心8分钟。

13)        用吸量管把上清转移到新的50ml的圆锥管中(上清1)。

14)        再往收集有细胞体的圆锥管中加入10ml的新鲜培养基,悬起细胞。

15)        再次在圆锥管架上震荡细胞,使细胞破碎。

16)        置于另一个较大的离心管中,以转速2000 rpm离心8分钟。

17)        用吸量管把上清转移到新的50ml的圆锥管中(上清2)。

18)        5ml的吸量管把上清12转移到小的冷冻管(CryoTube vials)中。(每管1ml)(管上标明Sup1还是 Sup 2以及IIIB、时间)

19)        保存在-80 °C冰箱。

 

配方:

 

1.       培养基 (500 mL)

RPMI1640培养基                      500 ml

FBS                                            50 ml

青霉素-链霉素                           5 ml

 

参考文献:

 

1.        Dutschman G.E., Grill S.P., Gullen E.A., Haraguchi K., Takeda S., Tanaka H., Baba M., Cheng Y.C. (2004). Novel 4'-substituted stavudine analog with improved anti-human immunodeficiency virus activity and decreased cytotoxicity. Antimicrobial Agents and Chemotherapy 48(5): 1640-6. 

 

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How to cite this protocol: Wang, X. (2012). Amplification of HIV-1 Infectious Virus in BL3 Lab. Bio-protocol 2(5): e102. DOI: 10.21769/BioProtoc.102; Full Text



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