欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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, 5375 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook