搜索

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines
A2和A72 J-Lat细胞系中药物诱导的HIV-1转录活性的流式细胞术分析   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Cell Cycle
Feb 2013

Abstract

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.

Keywords: Human immunodeficiency virus-1 (人体免疫缺陷病毒-1), Latency (潜伏期), Drug treatment (药物处理), Transcriptional activation (转录激活), HIV-1 LTR (HIV-1 LTR), Flow cytometry (流式细胞术), J-Lat cell lines (J-Lat细胞系)

Background

Studies that assess transcriptional activation or repression of the HIV-1 LTR generally use CD4+ T cells containing latent full-length HIV-1, such as NL4-3/E-/GFP-IRES–nef (Kutsch et al., 2002) or R7/E-/GFP (Jordan et al., 2003), which contains a frameshift mutation in the viral Env gene to prevent viral spread and expresses GFP in the Nef open reading frame allowing separation of actively infected GFP+ from GFP cells (uninfected or latently infected) by cell sorting (Jordan et al., 2003). To specifically investigate transcriptional activation of the HIV-1 LTR, we utilize the J-Lat cell line A72 containing only a latent LTR-GFP construct (Jordan et al., 2003). To determine if drug treatment specifically activates Tat, we utilize a J-Lat cell line harboring a latent lentiviral construct expressing Tat with GFP from the HIV-1 LTR (clone A2; LTR-Tat-IRES-GFP) (Jordan et al., 2003).

Materials and Reagents

  1. A2 and A72 J-Lat cell culture
    1. 75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353110 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. A2 and A72 J-Lat cells (Jordan et al., 2003)
    4. RPMI (Mediatech, catalog number: 10-040-CV )
    5. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    6. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    7. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )

  2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
    1. 96-well V-bottom tissue culture plates and lids (Thermo Fisher Scientific, Thermo Scientific TM, catalog numbers: N249570 and N163320 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. RPMI (Mediatech, catalog number: 10-040-CV )
    4. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    5. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    6. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    7. TNFα (PeproTech, catalog number: 300-01A )
    8. JQ1 (Cayman Chemical, catalog number: 11187 )
    9. Prostratin (Sigma-Aldrich, catalog number: P0077 )
    10. Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich, catalog number: SML0061 )
    11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    12. 1x PBS (Mediatech, catalog number: 21-031-CV )
    13. MACSQuant Running buffer (Miltenyi Biotech, catalog number: 130-092-747 )
    14. ApoTox-GloTM Triplex Assay (Promega, catalog number: G6320 )
    15. RPMI medium (see Recipes)
    16. TNFα stock solution (see Recipes)
    17. JQ1 stock solution (see Recipes)
    18. Prostratin stock solution (see Recipes)
    19. Suberoylanilide hydroxamic acid (SAHA) stock solution (see Recipes)

Equipment

  1. Pipette
  2. Biosafety cabinet level 2
  3. CO2 tissue culture incubator (Thermo Electron, model: FormaTM Steri-CultTM CO2 Incubators , catalog number: 3307)
  4. Tabletop centrifuge (Beckman Coulter, model: Allegra X-14R ) for 96-well plates
  5. MACSQuant VYB FACS analyzer (Miltenyi Biotech, model: MACSOuant® VYB, catalog number: 130-096-116 )
  6. SpectraMax MiniMaxTM 300 Imaging Cytometer (Molecular Devices, model: SpectraMax MiniMax 300 )

Software

  1. FlowJo 9.9 or never (Tree Star)

Procedure

  1. A2 and A72 J-Lat suspension cell culture
    1. A2 and A72 cells are cultured in RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) in 75 cm2 tissue culture flasks in 40 ml medium. The flasks are placed horizontally to increase the surface area in which the cells are grown.
    2. Cells are split every 1-2 days and should be kept at a concentration of 2 x 105/ml-1.5 x 106/ml.
    3. One day before drug treatment split A2 or A72 cells to 5 x 105/ml.

  2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
    1. Count the cells and adjust cell number with new media. About 2 x 105/well of A2 or A72 cells are plated in 96-well tissue culture plates in 195 µl RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin). For each condition perform three replicates.
    2. Cells are stimulated with 0.016-10 ng/ml of TNFα, 100 nM-1 µM JQ1, 100 nM-2.5 µM Prostratin, 330 nM-2.5 µM SAHA, DMSO control, or left untreated. If treated with TNFα, use PBS or water as control, depending on what was used as diluent. All drugs and controls are pipetted in 5 µl to reach a total volume of 200 µl/well. Mix cells and drugs by pipetting up and down at least 3 times.
    3. Culture in a CO2 incubator at 37 °C for 18-48 h.
    4. Take the plate from the incubator and centrifuge at room temperature for 2 min at 1341 x g (2,400 rpm) (Tabletop centrifuge, Beckman).
    5. Then proceed directly to flow cytometry analysis. Cells do not have to be washed or fixed.
      Notes:
      1. For flow cytometry, MACSQuant VYB FACS analyzer was used to run 96-well plates. However, other flow cytometers such as Calibur or LSRII are also suitable for these experiments.
      2. If the analysis of cell viability by forward scatter (FSC) vs. side scatter (SSC) is not sufficient for accurate assessment of drug toxicity in drug studies, one of a variety of dyes/stains; for example 7AAD, Propidium iodide or one of the Zombie viability dyes can be used according to manufacturer’s instructions in the flow cytometric analysis in this protocol.
    6. Viability, cytotoxicity and apoptosis was measured with ApoTox-GloTM Triplex Assay (Promega) according to manufacturer’s instructions using a SpectraMax MiniMaxTM 300 Imaging Cytometer (Figure 1).


      Figure 1. Measurement of Viability, Cytotoxicity and Apoptosis of drug treated cells. ApoTox-GloTM Triplex Assays (Promega) were performed in drug-treated A72 J-Lat cells. A. Cytotoxicity and Viability; B. Apoptosis; All measurements were repeated at least three times and average of three technical replicates (± SD) is shown.

Data analysis

Analysis of HIV-1 LTR transcriptional activation by flow cytometry (Figure 2)

  1. First, set the gate on live J-Lat cells. Cell viability is monitored by forward (FSC-Area) and side scatter (SSC-Area) analysis (Figure 2A).
  2. Gate on singlets (FSC-Height vs. FSC-Area) (Figure 2B).
  3. Set the gate on SSC-Area and GFP/FITC-Area to identify the amount of GFP+ cells (Figure 2C).
  4. Each sample is usually analyzed in triplicate and the experiment is performed with cells coming from at least 3 independent experiments (Figure 3). Three replicates are averaged by calculating (GFP+ cells Experiment 1 + GFP+ cells Experiment 2 + GFP+ cells Experiment 3)/3. Also calculate standard deviation (STDEV) for error bars.


    Figure 2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry. Gating strategy to analyze A2 or A72 J-Lat cells: A. gating on live J-Lat cells based on size (FSC-Area) and granularity (SSC-Area); B. singlets gate (FSC-Height vs. FSC-Area); C. gating on GFP+ J-Lat cells (SSC-Area vs. GFP-FITC-Area).


    Figure 3. HIV-1 LTR transcriptional activation by flow cytometry. Typical results obtained for 18 h treatment with TNFα, with dose dependent response using A2 J-Lat cells. Average for percentage of GFP+ cells from three replicates (± SD) is shown.

Recipes

  1. RPMI medium
    RPMI supplemented with,
    10% FBS
    1% L-glutamine
    1% penicillin/streptomycin
    Store at 4 °C
  2. TNFα stock solution
    100 ng/μl in sterile water
    Store at -80 °C
  3. JQ1 stock solution
    10 mM in DMSO
    Store at -80 °C
    Note: Avoid repeated freeze-thaws!
  4. Prostratin stock solution
    5 mM in sterile water
    Store at -20 °C
  5. Suberoylanilide hydroxamic acid (SAHA) stock solution
    10 mM in DMSO
    Store at -20 °C

Acknowledgments

We thank Marielle Cavrois and the Flow cytometry core for the service provided for flow cytometry. This publication was made possible with the help from the University of California, San Francisco–Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027763). This research was supported as part of the amfAR Institute for HIV Cure Research, with funding from amfAR grant number 109301. Further, we gratefully acknowledge support from the California HIV/AIDS Research Program (Award number: F13-GI-316) to D.B., and grant support from the CARE Collaboratory (U19 AI096113) and the NIH (RO1 AI083139 and RO1 DA043142) to M.O. This protocol was adapted from previous work: ‘BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism’ (Boehm et al., 2013).

References

  1. Archin, N. M., Espeseth, A., Parker, D., Cheema, M., Hazuda, D. and Margolis, D. M. (2009). Expression of latent HIV induced by the potent HDAC inhibitor suberoylanilide hydroxamic acid. AIDS Res Hum Retroviruses 25(2): 207-212.
  2. Boehm, D., Calvanese, V., Dar, R. D., Xing, S., Schroeder, S., Martins, L., Aull, K., Li, P. C., Planelles, V., Bradner, J. E., Zhou, M. M., Siliciano, R. F., Weinberger, L., Verdin, E. and Ott, M. (2013). BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism. Cell Cycle 12(3): 452-462.
  3. Contreras, X., Schweneker, M., Chen, C. S., McCune, J. M., Deeks, S. G., Martin, J. and Peterlin, B. M. (2009). Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells. J Biol Chem 284(11): 6782-6789.
  4. Delmore, J. E., Issa, G. C., Lemieux, M. E., Rahl, P. B., Shi, J., Jacobs, H. M., Kastritis, E., Gilpatrick, T., Paranal, R. M., Qi, J., Chesi, M., Schinzel, A. C., McKeown, M. R., Heffernan, T. P., Vakoc, C. R., Bergsagel, P. L., Ghobrial, I. M., Richardson, P. G., Young, R. A., Hahn, W. C., Anderson, K. C., Kung, A. L., Bradner, J. E. and Mitsiades, C. S. (2011). BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146(6): 904-917.
  5. Edelstein, L. C., Micheva-Viteva, S., Phelan, B. D. and Dougherty, J. P. (2009). Short communication: activation of latent HIV type 1 gene expression by suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor approved for use to treat cutaneous T cell lymphoma. AIDS Res Hum Retroviruses 25(9): 883-887.
  6. Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S. and Bradner, J. E. (2010). Selective inhibition of BET bromodomains. Nature 468(7327): 1067-1073.
  7. Finzi, D., Blankson, J., Siliciano, J. D., Margolick, J. B., Chadwick, K., Pierson, T., Smith, K., Lisziewicz, J., Lori, F., Flexner, C., Quinn, T. C., Chaisson, R. E., Rosenberg, E., Walker, B., Gange, S., Gallant, J. and Siliciano, R. F. (1999). Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5(5): 512-517.
  8. Geeraert, L., Kraus, G. and Pomerantz, R. J. (2008). Hide-and-seek: the challenge of viral persistence in HIV-1 infection. Annu Rev Med 59: 487-501.
  9. Jordan, A., Bisgrove, D. and Verdin, E. (2003). HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22(8): 1868-1877.
  10. Kelly, W. K., Richon, V. M., O'Connor, O., Curley, T., MacGregor-Curtelli, B., Tong, W., Klang, M., Schwartz, L., Richardson, S., Rosa, E., Drobnjak, M., Cordon-Cordo, C., Chiao, J. H., Rifkind, R., Marks, P. A. and Scher, H. (2003). Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 9(10 Pt 1): 3578-3588.
  11. Kutsch, O., Benveniste, E. N., Shaw, G. M. and Levy, D. N. (2002). Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. J Virol 76(17): 8776-8786.
  12. Quivy, V., Adam, E., Collette, Y., Demonte, D., Chariot, A., Vanhulle, C., Berkhout, B., Castellano, R., de Launoit, Y., Burny, A., Piette, J., Bours, V. and Van Lint, C. (2002). Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-kappaB and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies. J Virol 76(21): 11091-11103.
  13. Sedaghat, A. R., Siliciano, J. D., Brennan, T. P., Wilke, C. O. and Siliciano, R. F. (2007). Limits on replenishment of the resting CD4+ T cell reservoir for HIV in patients on HAART. PLoS Pathog 3(8): e122.
  14. Williams, S. A., Chen, L. F., Kwon, H., Fenard, D., Bisgrove, D., Verdin, E. and Greene, W. C. (2004). Prostratin antagonizes HIV latency by activating NF-κB. J Biol Chem 279(40): 42008-42017.

简介

消除HIV-1患者的主要障碍是后整合延迟(Finzi等,1999)。抗逆转录病毒治疗仅针对主动复制病毒,而具有低转录活性或无转录活性的潜伏性感染仍未得到治疗(Sedaghat et al。,2007)。抗逆转录病毒治疗与潜伏期清除策略的组合可以加速潜伏性储层的消耗并导致治愈(Geeraert等人,2008)。从潜伏期重新激活HIV-1的当前策略包括使用前列腺素,非促肿瘤佛波酯(Williams等人,2004),BET抑制剂(Filippakopoulos等人, (HDM)抑制剂,例如辛苯甲酰苯胺异羟肟酸(SAHA或Vorinostat))(Kelly ,2010; Delmore等人,2011)和组蛋白脱乙酰酶et al。,2003; Archin等人,2009; Contreras等人,2009; Edelstein等人。 ,2009)。由于HIV-1潜伏期的机制是多样的,有效的再激活可能需要组合策略(Quivy等人,2002)。以下方案描述了基于流式细胞仪的方法,用于量化药物治疗后HIV-1长末端重复(LTR)的转录激活。该协议被优化用于研究含有GFP盒的潜伏的HIV-1感染的Jurkat(J-Lat)细胞系。含有不同报道物的J-Lats,例如萤光素酶,可以用所述的药物进行治疗,但必须进行不同的分析。

背景 评估HIV-1 LTR的转录激活或抑制的研究通常使用含有潜在全长HIV-1(例如NL4-3 / E-GFP-IRES-nef)的CD4 + Kutsch等人,2002)或R7 / E- / GFP(Jordan等人,2003),其包含病毒环境中的移码突变,基因,以防止病毒扩散,并在Nef 开放阅读框中表达GFP,从而可以从GFP - 细胞分离积极感染的GFP + 未感染或潜伏感染)通过细胞分选(Jordan等人,2003)。为了特异性地研究HIV-1LTR的转录激活,我们利用仅含有潜在LTR-GFP构建体的J-Lat细胞系A72(Jordan等人,2003)。为了确定药物治疗是否特异性激活Tat,我们利用携带HIV-1 LTR(克隆A2; LTR-Tat-IRES-GFP)(Jordan等)的GFP表达Tat的潜伏性慢病毒构建物的J-Lat细胞系et。,2003)。

关键字:人体免疫缺陷病毒-1, 潜伏期, 药物处理, 转录激活, HIV-1 LTR, 流式细胞术, J-Lat细胞系

材料和试剂

  1. A2和A72 J-Lat细胞培养
    1. 75厘米组织培养瓶(Corning,Falcon ,目录号:353110)
    2. 提示
      0.1-10μl(Fisher Scientific,Fisherbrand TM,目录号:02-681-440)
      1-200μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-502)
      101-1,000μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-509)
    3. A2和A72 J-Lat细胞(Jordan等人,2003)
    4. RPMI(Mediatech,目录号:10-040-CV)
    5. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-106)
    6. L-谷氨酰胺(Mediatech,目录号:25-005-Cl)
    7. 100x青霉素/链霉素(Mediatech,目录号:30-002-Cl)

  2. 通过流式细胞术分析HIV-1 LTR转录激活
    1. 96孔V底组织培养板和盖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:N249570和N163320)
    2. 提示
      0.1-10μl(Fisher Scientific,Fisherbrand TM,目录号:02-681-440)
      1-200μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-502)
      101-1,000μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-509)
    3. RPMI(Mediatech,目录号:10-040-CV)
    4. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-106)
    5. L-谷氨酰胺(Mediatech,目录号:25-005-Cl)
    6. 100x青霉素/链霉素(Mediatech,目录号:30-002-Cl)
    7. TNFα(PeproTech,目录号:300-01A)
    8. JQ1(Cayman Chemical,目录号:11187)
    9. Prostratin(Sigma-Aldrich,目录号:P0077)
    10. 苯甲酰苯胺异羟肟酸(SAHA)(Sigma-Aldrich,目录号:SML0061)
    11. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
    12. 1x PBS(Mediatech,目录号:21-031-CV)
    13. MACSQuant运行缓冲区(Miltenyi Biotech,目录号:130-092-747)
    14. ApoTox-Glo TM Triplex Assay(Promega,目录号:G6320)
    15. RPMI培养基(见食谱)
    16. TNFα储备溶液(参见食谱)
    17. JQ1库存解决方案(见配方)
    18. Prostratin储备溶液(参见食谱)
    19. 苯乙酰苯胺异羟肟酸(SAHA)储备溶液(见配方)

设备

  1. 移液器
  2. 生物安全柜2级
  3. CO 2组织培养培养箱(Thermo Electron,型号:Forma< sup> Steri-Cult<> CO 2培养箱,目录号码:3307)
  4. 用于96孔板的台式离心机(Beckman Coulter,型号:Allegra X-14R)
  5. MACSQuant VYB FACS分析仪(Miltenyi Biotech,型号:MACSOuant ® VYB,目录号:130-096-116)
  6. SpectraMax MiniMax TM 300成像细胞仪(Molecular Devices,型号:SpectraMax MiniMax 300)

软件

  1. FlowJo 9.9或永不(Tree Star)

程序

  1. A2和A72 J-Lat悬浮细胞培养
    1. 将A2和A72细胞在40ml培养基中的75cm 2组织培养瓶中的RPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)中培养。将烧瓶水平放置以增加细胞生长的表面积。
    2. 细胞每1-2天分裂一次,并应保持浓度为2×10 5/ml〜1.5×10 -6/ml。
    3. 药物治疗前一天将A2或A72细胞分裂至5×10 5/ml。

  2. 通过流式细胞术分析HIV-1 LTR转录激活
    1. 计数单元格并用新介质调整单元格编号。将约2×10 5个/孔的A2或A72细胞铺在96孔组织培养板中的195μlRPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)。每个条件执行三次重复。
    2. 用0.016-10ng/mlTNFα,100nM-1μMJQ1,100nM-2.5μMProstratin,330nM-2.5μMSAHA,DMSO对照刺激细胞,或未处理。如果用TNFα处理,使用PBS或水作为对照,取决于用作稀释剂。所有药物和对照物以5μl的量移液,达到200μl/孔的总体积。通过上下移动将细胞和药物混合至少3次。
    3. 在CO 2培养箱中37℃培养18-48小时。
    4. 从培养箱取板,并在室温下以1341 x g(2,400 rpm)(台式离心机,Beckman)离心2分钟。
    5. 然后直接进行流式细胞分析。细胞不必被洗涤或固定。
      注意:
      1. 对于流式细胞术,使用MACSQuant VYB FACS分析仪来运行96孔板。然而,其他流式细胞仪如Calibur或LSRII也适用于这些实验。
      2. 如果通过前向散射(FSC)与侧向散射(SSC)的细胞活力的分析不足以准确评估药物研究中的药物毒性,则是各种染料/污渍之一;例如7AAD,碘化丙啶或其中一种Zombie活性染料可以按照本协议流程细胞分析中的制造商说明使用。
    6. 根据制造商的说明书,使用SpectraMax MiniMax TM/300多成像细胞仪(图1),用ApoTox-Glo Triplex Assay(Promega)测量活力,细胞毒性和细胞凋亡。 />

      图1.药物治疗细胞的活力,细胞毒性和细胞凋亡的测量。在药物处理的A72 J-Lat细胞中进行ApoTox-GloTM Triplex测定(Promega)。 A.细胞毒性和活力; B.细胞凋亡;所有测量重复至少三次,平均显示三次技术重复(±SD)。

数据分析

通过流式细胞术分析HIV-1 LTR转录激活(图2)

  1. 首先,在活的J-Lat细胞上设置门。通过正向(FSC区域)和侧向散射(SSC-Area)分析(图2A)监测细胞活力。
  2. 单刀闸(FSC-Height vs. FSC-Area)(图2B)
  3. 在SSC-Area和GFP/FITC-Area上设置门,以识别GFP + 单元格的数量(图2C)。
  4. 每个样品通常一式三份分析,实验用来自至少3次独立实验的细胞进行(图3)。通过计算(GFP + 细胞实验1 + GFP + 细胞实验2 + GFP + 细胞实验3)/3来平均三个重复。还计算误差条的标准偏差(STDEV)。


    图2.通过流式细胞术分析HIV-1 LTR转录激活
    分析A2或A72 J-Lat细胞的门控策略:A.基于大小(FSC区域)对活J-Lat细胞门控)和粒度(SSC-Area); B.单闸门(FSC-Height vs. FSC-Area); C.Gate on GFP + J-Lat细胞(SSC-Area vs.GFP-FITC-Area)。


    图3.流式细胞术检测HIV-1 LTR转录激活。 用TNFα处理18小时的典型结果,使用A2 J-Lat细胞剂量依赖性反应。显示来自三个重复(±SD)的GFP + 细胞百分比的平均值。

食谱

  1. RPMI媒体
    RPMI补充了,
    10%FBS
    1%L-谷氨酰胺
    1%青霉素/链霉素
    储存于4°C
  2. TNFα储备液
    100 ng /μl,无菌水 储存于-80°C
  3. JQ1库存解决方案
    10mM在DMSO中 储存于-80°C
    注意:避免反复冻融!
  4. Prostratin储备溶液
    5毫升无菌水
    储存于-20°C
  5. 苯甲酰苯胺异羟肟酸(SAHA)储备溶液
    10mM在DMSO中 储存于-20°C

致谢

我们感谢Marielle Cavrois和流式细胞术核心为流式细胞术提供的服务。加利福尼亚大学旧金山Gladstone病毒学研究所的帮助下,本出版物成为可能。艾滋病研究免疫中心(CFAR),NIH资助计划(P30 AI027763)。这项研究得到了amfAR艾滋病毒治疗研究所的一部分的支持,该研究所获得了来自amfAR拨款109301的资助。此外,我们非常感谢加州艾滋病研究项目(F13-GI-316)获得DB支持,并向CARE协作机构(U19 AI096113)和NIH(RO1 AI083139和RO1 DA043142)授予MO支持该协议改编自以前的工作:"BET溴结构域靶向化合物通过Tat独立机制从延迟重新激活HIV"(Boehm等人,2013)。

参考

  1. Archin,NM,Espeseth,A.,Parker,D.,Cheema,M.,Hazuda,D.and Margolis,DM(2009)。  由有效的HDAC抑制剂辛二酰苯胺异羟肟酸诱导的潜伏性HIV的表达。艾滋病Res Hum逆转录病毒 25 2):207-212。
  2. Boehm,D.,Calvanese,V.,Dar,RD,Xing,S.,Schroeder,S.,Martins,L.,Aull,K.,Li,PC,Planelles,V.,Bradner,JE,Zhou,MM ,Siliciano,RF,Weinberger,L.,Verdin,E.和Ott,M。(2013)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/23255218"target ="_ blank"> BET溴结构域靶向化合物通过Tat无关机制从延迟中重新激活HIV。细胞周期 12(3):452-462。
  3. Contreras,X.,Schweneker,M.,Chen,CS,McCune,JM,Deeks,SG,Martin,J.and Peterlin,BM(2009)。< a class ="ke-insertfile"href ="http: //www.ncbi.nlm.nih.gov/pubmed/19136668"target ="_ blank"> Suberoylanilide异羟肟酸重新激活来自潜在感染细胞的HIV。 J Biol Chem 284(11) :6782-6789。
  4. Delmore,JE,Issa,GC,Lemieux,ME,Rahl,PB,Shi,J.,Jacobs,HM,Kastritis,E.,Gilpatrick,T.,Paranal,RM,Qi,J.,Chesi,M.,Schinzel ,AC,McKeown,MR,Heffernan,TP,Vakoc,CR,Bergsagel,PL,Ghobrial,IM,Richardson,PG,Young,RA,Hahn,WC,Anderson,KC,Kung,AL,Bradner,JE和Mitsiades,CS (2011)。 BET溴结构域抑制作为一种治疗策略目标c-Myc。 细胞 146(6):904-917。
  5. Edelstein,LC,Micheva-Viteva,S.,Phelan,BD和Dougherty,JP(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19689202"target ="_ blank">短暂沟通:通过辛伐苯酰苯胺异羟肟酸(SAHA)激活潜在的HIV 1型基因表达,其是批准用于治疗皮肤T细胞淋巴瘤的HDAC抑制剂。 Res Hum RetroViruses 25(9):883-887。
  6. Filippakopoulos,P.,Qi,J.,Picaud,S.,Shen,Y.,Smith,WB,Fedorov,O.,Morse,EM,Keates,T.,Hickman,TT,Felletar,I.,Philpott,M ,Munro,S.,McKeown,MR,Wang,Y.,Christie,AL,West,N.,Cameron,MJ,Schwartz,B.,Heightman,TD,La Thangue,N.,French,CA,Wiest, O.,Kung,AL,Knapp,S.and Bradner,JE(2010)。  选择性抑制BET溴结构域。自然 468(7327):1067-1073。
  7. Finzi,D.,Blankson,J.,Siliciano,JD,Margolick,JB,Chadwick,K.,Pierson,T.,Smith,K.,Lisziewicz,J.,Lori,F.,Flexner,C.,Quinn, TC,Chaisson,RE,Rosenberg,E.,Walker,B.,Gange,S.,Gallant,J.and Siliciano,RF(1999)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/10229227"target ="_ blank"> CD4 + T细胞的潜在感染为HIV-1的终身持续存在提供了机制,即使在有效的联合治疗。 Nat Med 5(5):512-517。
  8. Geeraert,L.,Kraus,G.and Pomerantz,RJ(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17845138"target ="_ blank">隐藏寻求:HIV-1感染中病毒性持续存在的挑战。 Annu Rev Med 59:487-501。
  9. 约旦,A.,Bisgrove,D.和Verdin,E.(2003)。  HIV可重复地在体外T细胞急性感染后建立潜伏感染。 22(8):1868-1877。
  10. Kelly,WK,Richon,VM,O'Connor,O.,Curley,T.,MacGregor-Curtelli,B.,Tong,W.,Klang,M.,Schwartz,L.,Richardson,S.,Rosa,E 。,Drobnjak,M.,Cordon-Cordo,C.,Chiao,JH,Rifkind,R.,Marks,PA and Scher,H。(2003)。  组蛋白脱乙酰酶抑制剂的一期临床试验:静脉内施用的辛二酰苯胺异羟肟酸。 em> 9(10 Pt 1):3578-3588。
  11. Kutsch,O.,Benveniste,EN,Shaw,GM and Levy,DN(2002)。  直接和定量的单细胞分析人类免疫缺陷病毒1型从潜伏期重新激活。 76(17):8776-8786。
  12. Quivy,V.,Adam,E.,Collette,Y.,Demonte,D.,Chariot,A.,Vanhulle,C.,Berkhout,B.,Castellano,R.,de Launoit,Y.,Burny, ,Piette,J.,Bours,V.and Van Lint,C.(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12368351"target ="_ blank"> NF-κB和脱乙酰酶抑制剂对人类免疫缺陷病毒1型启动子活性的协同激活:制定治疗策略的潜在观点。 (21):11091-11103。
  13. Sedaghat,AR,Siliciano,JD,Brennan,TP,Wilke,CO和Siliciano,RF(2007)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17784786"target ="_ blank">在HAART患者中用于HIV的静息CD4 + T细胞库的补充限制 PLoS Pathog 3 (8):e122。
  14. Williams,SA,Chen,LF,Kwon,H.,Fenard,D.,Bisgrove,D.,Verdin,E.and Greene,WC(2004)。  Prostratin通过激活NF-κB拮抗HIV潜伏期。 J Biol Chem 279(40 ):42008-42017。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Boehm, D. and Ott, M. (2017). Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines. Bio-protocol 7(10): e2290. DOI: 10.21769/BioProtoc.2290.
提问与回复

(提问前,请先登录)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,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。