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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, 6559 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] 该方法用于制备高滴度人免疫缺陷病毒1型(HIV-1)病毒原液用于随后的感染测定。 T向性HIV-1病毒(IIIB株)的扩增使用CD4 + T细胞系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. CD4 + T细胞系H9细胞(ATCC HTB-176)
  2. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:SH30071.03 HI)
    注意:这个特定的FBS已经由作者测试,但可以由用户期望的来自不同供应商的FBS替代。
  3. 青霉素 - 链霉素液体(Life Technologies,Gibco ,目录号:15070-063)
  4. 细胞培养基:RPMI1640(Life Technologies,Gibco ,目录号:11875-093)(参见配方)

设备

  1. 台式离心机
  2. CO <2>孵化器
  3. -80°C冰箱
  4. 50ml锥形管(BD Biosciences,Falcon ,目录号:35-2070)
  5. (BD Biosciences,Falcon ,目录号:35-7501,35-7554,35-7556),在37℃,5分钟,10分钟,
  6. T-75,T-175细胞培养瓶(BD Biosciences,Falcon ,目录号:35-3136,35-3112)
  7. CryoTube小瓶(NUNC,377267)

程序

第1天

  1. 在50ml的falcon管中,在2ml培养基(终浓度1×10 6个细胞/ml)中稀释2×10 6个未感染的H9细胞。
  2. 向Falcon管中加入1ml HIV-1病毒(IIIB菌株)。
  3. 每15分钟轻轻摇动1小时。
  4. 将这种最低限度的培养物吸入50 ml培养基中的T-75细胞培养瓶中
  5. 在37℃孵育4天 第5天
  6. 向2个新的大型隼T-175细胞培养瓶中的每一个中加入175ml新鲜培养基
  7. 向每个烧瓶中加入25ml的4天培养物。
  8. 在37℃孵育3天 第8天
  9. 使用25-ml移液管(每管40ml)将HIV-1感染的细胞(400ml)转移到10×50-ml falcon管中。
  10. 在二级容器中以1300rpm离心8分钟。
  11. 使用真空管小心地移除上清液,用移液管吸头连接到10ml移液管(填充收集瓶用漂白剂)。
  12. 向每个管中加入2ml新鲜培养基并松开细胞沉淀。
  13. 收集所有细胞在2管中,用新鲜的培养基冲洗其他8管,并添加到那些2管。
  14. 向这两个管中的每一个中加入新鲜培养基至终体积为40ml。
  15. 在二级容器中以1300rpm离心8分钟。
  16. 除去上清液。
  17. 向每个烧瓶中加入10ml新鲜培养基,并松开细胞沉淀
  18. 组合成一个管。
  19. 使用管架击打细胞20秒。
  20. 在二级容器中以2,000rpm离心8分钟。
  21. 使用移液管转移上清液到新鲜的50毫升falcon管(上清液1)。
  22. 加入10毫升新鲜培养基与细胞沉淀管和松动细胞沉淀。
  23. 使用管架再次击打细胞。
  24. 在二级容器中以2,000rpm离心8分钟。
  25. 使用移液管转移上清液到新鲜的50ml的falcon管(上清液2)。
  26. 使用5ml移液管(每管1ml)(具有IIIB的标签管,日期和Sup1或Sup2)将Tranfer上清液1和2倒入小CryoTube小瓶中。
  27. 储存于-80°C冰箱

笔记

使用HIV-1的实验需要在BSL-3实验室进行,人员需要接受针对HIV-1病毒的特殊培训。

食谱

  1. 培养基(500ml)
    RPMI1640介质
    500 ml
    FBS
    50 ml
    青霉素 - 链霉素
    5 ml

致谢

作者感谢Dutschman,GE和Yung-Chi Cheng在耶鲁医学院药理学系的适应这种方法和他们对抗HIV-1药物开发研究的支持。

参考文献

  1. Dutschman,G.E.,Grill,S.P.,Gullen,E.A.,Haraguchi,K.,Takeda,S.,Tanaka,H.,Baba,M.and Cheng,Y.C。(2004)。 小说4'-取代的司他夫定 类似物具有改善的抗人类免疫缺陷病毒活性和降低的细胞毒性。 Antimicrob Agents Chemother 48(5):1640-1646。
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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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