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HIV-1 Fusion Assay
HIV-1 细胞融合分析   

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Abstract

The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

Materials and Reagents

  1. 293T cells (ATCC)
  2. pAdVAntage (Promega Corporation, catalog number: E1711 )
  3. pCMV4-BlaM-Vpr (Addgene, catalog number: 21950 )
  4. pNL4-3 proviral DNA (NIH AIDS Reagent Program) or TN6-GFP encoding primary Env (available upon request to Warner Greene)
  5. DMEM (Mediatech, Cellgro®, catalog number: 10-013-CV )
  6. RPMI 1640 (Mediatech, Cellgro®, catalog number: 15-040-CV )
  7. 1x phosphate-buffered saline (PBS)
  8. Fetal bovine serum (FBS)
  9. 100 U/ml of penicillin and 100 U/ml of streptomycin (Life Technologies, Gibco® catalog number: 15140-122 )
  10. 2 M CaCl2
  11. Alliance HIV-I p24 ELISA kit (PerkinElmer, catalog number: NEK050B001KT ) or FlaQ assay reagents (Hayden et al., 2003)
  12. Peripheral blood lymphocytes (PBLs)
  13. CCF2-AM substrate and loading solutions (Life Technologies, catalog number: K1032 )
  14. CO2-independent media (Life Technologies, Gibco®, catalog number: 18045-088 )
  15. Probenecid (Sigma-Aldrich, catalog number: P8761 )
  16. Mouse antihuman CD3 conjugated to APC-Cy7 and mouse antihuman CD4 conjugated to PE-Cy7 (BD Biosciences, catalog numbers: 557832 and 557852 )
  17. BD CompBeads (BD Biosciences, catalog number: 552843 )
  18. 16% Paraformalehyde (Electron Microscopy Sciences, catalog number: 15710 )
  19. HBSS (see Recipes)
  20. Dulbecco’s modified Eagle medium (DMEM) culture media (see Recipes)
  21. Roswell Park Memorial Institute (RPMI) culture media (see Recipes)
  22. CCF2 loading solution (see Recipes)
  23. Stock solution of probenecid (250 mM) prepared in 250 mM NaOH (see Recipes)
  24. Development media (see Recipes)

Equipment

  1. T175 cm2 culture flasks
  2. 96-well V-bottom plate (Corning Incorporated, catalog number: 3363 )
  3. 5, 10, 25 ml pipettes
  4. 50-ml Falcon tube
  5. 0.22 μm poresize Steriflip (EMD Millipore, catalog number: SE1M179M6 )
  6. 2 ml Nalgene tubes (Thermo Fisher Scientific, Nalgene®, catalog number: 5000-0020 )
  7. Clear ultracentrifuge tubes (BD, catalog number: BD340437 )
  8. 37 °C, 5% CO2 incubator set at 90% humidity
  9. Ultracentrifugation equipment with SW28 rotor
  10. Flow cytometer
    Note: The fusion assay alone requires a flow cytometer equipped with a violet laser excitation (405 nm) and two measurement parameters. The photomultiplicator tube (PMT) with a 450/50 nm band pass filter, commonly used for the detection of Pacific Blue, is used for the detection of the cleaved CCF2 substrate. The other PMT with a 515/20 nm band pass filter, commonly used for Amcyan detection, is used for the detection of the uncleaved CCF2 substrate. Additional PMTs are necessary for the measurement of the fluorochromes associated with the CD3 and CD4 antibodies. APC-Cy7 is excited by 633 nm red laser and detected with a PMT with a 755 long pass filters. PE-Cy7 is excited by a 531 nm yellow-green laser and detected with the PMT with a 755 long pass filter.

Software

  1. FlowJoX software (Tree Star) or other FACS analysis software

Procedure

  1. Plate 1.5 x 107 293T cells in a T175-cm2 tissue culture flask with 20 ml of DMEM culture media and culture overnight at 37 °C in a 5% CO2 humidified incubator.
  2. Prepare 1.75 ml of H2O containing 60 μg of TN6-GFP proviral DNA, 20 μg of pCMV-BlaM-Vpr, and 10 μg of pAdVAntage vectors.
  3. Slowly add 2 ml of 2x HBSS and mix gently by pipetting up and down.
  4. Add 250 μl of 2 M CaCl2 drop by drop.
  5. Incubate 10 min at room temperature to precipitate the DNA.
  6. Replace the 293T cell culture media with 40 ml of fresh DMEM culture media pre-warmed to 37 °C.
  7. Slowly add 4 ml of DNA precipitate and incubate for 16 h at 37 °C.
  8. Replace the media with 40 ml of fresh DMEM culture media pre-warmed to 37 °C.
  9. Incubate for 24 h at 37 °C.
  10. Harvest the supernatant of the transfected 293T cells into a 50-ml Falcon tube.
  11. Centrifuge the supernatant at 365 x g at room temperature for 10 min to remove the cellular debris.
  12. Filter the clarified supernatant through Steriflip.
  13. Transfer 36 ml of virion-containing supernatant to Ultra-Clear centrifuge tubes.
  14. Place the tube in the bucket of the SW28 rotor and balance the tube with DMEM culture media if necessary.
  15. Ultracentrifuge (72,000 x g, 90 min) at 4 °C without using brakes.
  16. Remove the supernatant, resuspend the viral pellet in 1 ml of DMEM, divide into 100-μl aliquots, and store at -80 °C.
  17. Quantify p24Gag content of the viral preparation by Enzyme-Linked Immunosorbent Assay or FlaQ Assay (Maiti et al., 2014).
  18. Wash the PBLs with RPMI culture media.
  19. Count PBLs and make a suspension 2 x 107 cells/ml in RPMI culture media.
  20. Divide the cell suspension in aliquots of 100 μl (2 x 106 cells) per condition to be tested in a V-bottom 96-well plate. Distribute two additional aliquots of cells into two wells to serve as compensation controls. One well will be loaded with CCF2 substrate while the other will remain unloaded. These two control wells will not be stained with anti-CD3-APC-Cy7 and anti-CD4-PE-Cy7 antibodies.
  21. Add a quantity of HIV-1 virions containing BlaM-Vpr equivalent to 400 ng of p24Gag to all wells except the “non infected control” and the two compensation controls. Incubate for 2 h at 37 °C.
  22. Collect the cells by centrifugation at 365 x g for 5 min at room temperature.
  23. Wash the cells once with 200 μl of CO2-independent media and centrifuge at 365 x g for 5 min at room temperature.
  24. Resuspend the pellet in 100 μl of CCF2-AM loading solution and incubate for 1 h at room temperature in the dark. Make sure to keep one well unstained by resuspending with CO2 independent media only.
  25. Collect the cells by centrifugation at 365 x g for 5 min at room temperature.
  26. Wash the cells with 200 μl of development media and centrifuge at 365 x g for 5 min at room temperature.
  27. Resuspend the pellet in 200 μl of development media and incubate the cells at room temperature for 16 h in the dark.
  28. In two empty wells, add one drop of each of BD compensation beads vials (negative control and Anti-mouse IgK).
  29. Collect the cells and beads by centrifugation at 365 x g for 5 min at 4 °C.
  30. Wash the cells and beads once by addition of 200 μl of FACS staining buffer.
  31. Collect the cells by centrifugation at 365 x g for 5 min at 4 °C and resuspend the pellet in 100 μl of immunostaining solution (in FACS staining buffer) containing a 1/100 dilution of anti-CD3-APC-Cy7 and a 1/50 dilution of anti-CD4-PE-Cy7. Do not stain the 2 compensation controls nor the beads. Stain the CompBeads with a FACS staining buffer containing either a 1/100 dilution of anti-CD3-APC, or a 1/50 dilution of anti-CD4-PE-Cy7.
  32. Incubate for 30 min at 4 °C.
  33. Collect the cells and beads by centrifugation at 365 x g for 5 min at 4 °C.
  34. Wash the cells and beads with 200 μl of FACS staining buffer.
  35. Fix the cells in FACS staining buffer supplemented with 1.2% paraformaldehyde for 24 h at 4 °C.
  36. Acquire the samples on a FACS instrument. The set of samples should include: unloaded unstained cells, the CCF2 loaded unstained cells, the CD3-APC-Cy7 stained beads, the CD4-PE-Cy7 stained beads, the non-infected control loaded with CCF2 and immunostained, and the infected samples loaded with CCF2 and immunostained. Make sure to acquire emission in the FITC channel in addition to the parameters of the assay (uncleaved and cleaved substrate, APC-Cy7 and PE-Cy7). The voltage should be set using the non-infected, immunostained sample.
  37. Import the fcs files into the FlowJo software. Use the fcs files corresponding to the CompBeads to compensate for APC-Cy7 and PE-Cy7, use the CCF2 loaded and unloaded samples to compensate in the FITC channel. Apply the compensation to all samples.
  38. Gate the cells as shown Figure 2.

Representative data



Figure 1. Principle of the fusion assay


Figure 2. Representative data. A. Gating strategy to measure HIV-1 fusion to resting PBL cultures. The population of cells was first gated on Forward Scatter area (FSC-A) and Side Scatter area (SSC-A). Single cells were then gated on a FACS plot with FSC-A and FSC-H. The population of CD4 T cells was gated on APC-Cy7 and PE-Cy7. The fusion gate was drawn on the non infected sample in a FACS plot representing cleaved CCF2 versus uncleaved CCF2. B. Representative fusion FACS plot obtained for non-infected sample (NI), the X4-tropic NL4-3, the R5-tropic 81A and a primary HIV-1 envelope (Env) subcloned into TN6-GFP (Cavrois et al., 2011; Cavrois et al., 2014).

Notes

  1. While there is donor-to-donor variability in the susceptibility to HIV-1 fusion, normalization by 81A shows very good assessment of the relative ability of a given primary Env to mediate fusion (Cavrois et al., 2014).

Recipes

  1. HBSS
    280 mM NaCl
    10 mM KCl
    1.5 mM Na2HPO4
    12 mM dextrose
    50 mM N-(2-hydroxyethylpiperazine)-N′-(2-ethanesulfonic acid) (HEPES) (pH 7.05)
    Stored at -20 °C
  2. Fluorescence-activated cell sorting (FACS) staining buffer
    1x phosphate-buffered saline (PBS)
    2% fetal bovine serum
    Stored at 4 °C
  3. Dulbecco’s modified Eagle medium (DMEM) culture media
    DMEM
    10% fetal bovine serum
    100 U/ml of penicillin
    100 μg/ml of streptomycin
  4. RPMI culture media
    RPMI 1640
    10% fetal bovine serum
    100 U/ml of penicillin
    100 μg/ml of streptomycin
  5. CCF2 loading solution
    1. Resuspend CCF2-AM in dimethylsulfoxide (solution A of the CCF2 kit) to generate a stock solution (1 mM CCF2-AM). Divided into aliquots and stored in the dark at -80 °C.
    2. Mix by vortex-mixing 1 μl of 1 mM CCF2-AM with 9 μl of a solution containing 100 mg/ml of Pluronic-F127 and 0.1% acetic acid (solution B of the CCF2 kit).
    3. Add 1 ml of CO2-independent media and vortex again.
  6. Stock solution of probenecid (250 mM) prepared in 250 mM NaOH
    Divided into aliquots and store at -20 °C
  7. Development media
    2.5 mM probenecid
    10% fetal bovine serum in CO2-independent media

Acknowledgments

This assay was first published in (Cavrois et al., 2002) and the protocol described in detail in (Cavrois et al., 2004). The fusion assay also allows studies of fusion mediated by other viruses, including Ebola (Yonezawa et al., 2005) and henipavirus (Wolf et al., 2009). We would like to thank the team lead by Prof. Tsien for developing CCF2, the BlaM substrate (Zlokarnik et al., 1998).

References

  1. Cavrois, M., De Noronha, C. and Greene, W. C. (2002). A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20(11): 1151-1154.
  2. Cavrois, M., Neidleman, J., Bigos, M. and Greene, W. C. (2004). Fluorescence resonance energy transfer-based HIV-1 virion fusion assay. Methods Mol Biol 263: 333-344.
  3. Cavrois, M., Neidleman, J., Galloway, N., Derdeyn, C. A., Hunter, E. and Greene, W. C. (2011). Measuring HIV fusion mediated by envelopes from primary viral isolates. Methods 53(1): 34-38.
  4. Cavrois, M., Neidleman, J., Santiago, M. L., Derdeyn, C. A., Hunter, E. and Greene, W. C. (2014). Enhanced fusion and virion incorporation for HIV-1 subtype C envelope glycoproteins with compact V1/V2 domains. J Virol 88(4): 2083-2094.
  5. Hayden, M. S., Palacios, E. H. and Grant, R. M. (2003). Real-time quantitation of HIV-1 p24 and SIV p27 using fluorescence-linked antigen quantification assays. AIDS 17(4): 629-631. 
  6. Maiti, M., Grant, R. and Cavrois, M. (2014). Fluorescence-linked antigen quantification (FLAQ) assay for fast quantification of HIV-1 p24Gag. Bio-protocol 4(24): e1366.
  7. Wolf, M. C., Wang, Y., Freiberg, A. N., Aguilar, H. C., Holbrook, M. R. and Lee, B. (2009). A catalytically and genetically optimized beta-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics. Virol J 6: 119.
  8. Yonezawa, A., Cavrois, M. and Greene, W. C. (2005). Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha. J Virol 79(2): 918-926.
  9. Zlokarnik, G., Negulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. and Tsien, R. Y. (1998). Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279(5347): 84-88.

简介

HIV-1融合测定测量HIV-1生命周期中直至并包括病毒融合的所有步骤。 它依赖于将β-内酰胺酶-Vpr(BlaM-Vpr)蛋白嵌合体并入病毒体中,并且随后通过融合将该嵌合体转移到靶细胞中(图1)。 通过CCF2(一种β-内酰胺酶的荧光染料底物)的酶裂解来监测转移,将其装载到靶细胞中。 β-内酰胺酶在CCF2中的β-内酰胺环的切割将染料的荧光发射光谱从绿色(520nm)改变为蓝色(447nm)。 这种变化反映了病毒体融合,并且可以通过流式细胞术检测(图2)。

材料和试剂

  1. 293T细胞(ATCC)
  2. pAdVAntage(Promega Corporation,目录号:E1711)
  3. pCMV4-BlaM-Vpr(Addgene,目录号:21950)
  4. pNL4-3原病毒DNA(NIH AIDS试剂程序)或TN6-GFP编码初级Env(可根据要求提供给Warner Greene)
  5. DMEM(Mediatech,Cellgro ,目录号:10-013-CV)
  6. RPMI 1640(Mediatech,Cellgro ,目录号:15-040-CV)
  7. 1×磷酸盐缓冲盐水(PBS)
  8. 胎牛血清(FBS)
  9. 100 U/ml青霉素和100U/ml链霉素(Life Technologies,Gibco 目录号:15140-122)。
  10. 2 M CaCl 2
  11. Alliance HIV-1 p24 ELISA试剂盒(PerkinElmer,目录号:NEK050B001KT)或FlaQ测定试剂(Hayden等人,2003)
  12. 外周血淋巴细胞(PBL)
  13. CCF2-AM底物和上样溶液(Life Technologies,目录号:K1032)
  14. CO 2独立培养基(Life Technologies,Gibco ,目录号:18045-088)。
  15. 丙磺舒(Sigma-Aldrich,目录号:P8761)
  16. 偶联到APC-Cy7的小鼠抗人CD3和偶联到PE-Cy7的小鼠抗人CD4(BD Biosciences,目录号:557832和557852)
  17. BD CompBeads(BD Biosciences,目录号:552843)
  18. 16%Paraformalehyde(Electron Microscopy Sciences,目录号:15710)
  19. HBSS(参见配方)
  20. Dulbecco改良的Eagle培养基(DMEM)培养基(参见Recipes)
  21. 罗斯威尔公园纪念研究所(RPMI)文化媒体(参见食谱)
  22. CCF2加载解决方案(参见配方)
  23. 在250mM NaOH中制备的丙磺舒(250mM)储备溶液(参见Recipes)
  24. 开发媒体(见配方)

设备

  1. T175cm 2培养瓶中
  2. 96孔V型底板(Corning Incorporated,目录号:3363)
  3. 5,10,25 ml移液器
  4. 50 ml Falcon管
  5. 0.22μm孔化Steriflip(EMD Millipore,目录号:SE1M179M6)
  6. 2ml Nalgene管(Thermo Fisher Scientific,Nalgene ,目录号:5000-0020)
  7. 清除超高速离心管(BD,目录号:BD340437)
  8. 37℃,设定在90%湿度的5%CO 2培养箱中
  9. 带SW28转子的超速离心设备
  10. 流式细胞仪
    注意:单独的融合测定需要配备有紫激光激发(405nm)和两个测量参数的流式细胞仪。具有通常用于检测太平洋蓝的450/50nm带通滤波器的光电倍增管(PMT)用于检测裂解的CCF2衬底。具有515/20nm带通滤波器(通常用于Amcyan检测)的另一PMT用于检测未裂解的CCF2底物。另外的PMT是测量与CD3和CD4抗体相关的荧光染料所必需的。 APC-Cy7由633nm红色激光激发,并用具有755长通滤波器的PMT检测。 PE-Cy7由531nm黄绿色激光激发并用具有755长通滤光片的PMT检测。

软件

  1. FlowJoX软件(Tree Star)或其他FACS分析软件

程序

  1. 在具有20ml DMEM培养基的T175-cm 2组织培养烧瓶中铺板1.5×10 7个293T细胞并在37℃在5%CO 2中培养过夜, sub> 2 加湿培养箱
  2. 制备含有60μgTN6-GFP前病毒DNA,20μgpCMV-BlaM-Vpr和10μgpAdVAntage载体的1.75ml H 2 O O.
  3. 缓慢加入2ml 2×HBSS,并通过上下吹打轻轻混匀
  4. 逐滴加入250μl的2M CaCl 2。
  5. 在室温孵育10分钟以沉淀DNA。
  6. 用预先温热至37℃的40ml新鲜DMEM培养基替换293T细胞培养基
  7. 缓慢加入4ml DNA沉淀,并在37℃下孵育16小时
  8. 用40毫升预热至37℃的新鲜DMEM培养基更换培养基
  9. 在37℃下孵育24小时
  10. 将转染的293T细胞的上清液收获到50-ml Falcon管中
  11. 在365离心上清液在室温下10分钟,以去除细胞碎片。
  12. 通过Steriflip过滤澄清的上清液
  13. 将36ml含有病毒粒子的上清液转移到Ultra-Clear离心管中
  14. 将管放置在SW28转子的桶中,如有必要,用DMEM培养基平衡管。
  15. 在4℃,不使用制动器的超速离心机(72,000×g ,90分钟)。
  16. 除去上清液,将病毒沉淀重悬在1ml DMEM中,分成100μl等分试样,并储存在-80℃。
  17. 通过酶联免疫吸附测定或FlaQ测定来定量病毒制备物的p24 Gag 含量(Maiti等人,2014)。
  18. 用RPMI培养基洗涤PBL。
  19. 计数PBL,并在RPMI培养基中制成悬浮液2×10 7细胞/ml。
  20. 将细胞悬浮液在每个待测试条件的100μl(2×10 6个细胞)的等分试样中在V型底96孔板中分开。 将两个另外的细胞等分试样分配到两个孔中作为补偿对照。 一个井将装载CCF2基板,而另一个井将保持未装载。 这两个对照孔不用抗CD3-APC-Cy7和抗CD4-PE-Cy7抗体染色。
  21. 向除了"非感染对照"和两个补偿对照之外的所有孔中加入一定量的含有等于400ng p24Gag 的BlaM-Vpr的HIV-1病毒颗粒。在37℃下孵育2小时。
  22. 通过在365℃下离心5分钟,在室温下收集细胞5分钟
  23. 用200μl的CO 2非依赖性培养基洗涤细胞一次,并在室温下以365×g离心5分钟。
  24. 将沉淀重悬在100μl的CCF2-AM加载溶液中,并在室温下在黑暗中孵育1小时。确保通过与CO 2独立介质重新悬浮保持一个未污染。
  25. 通过在365℃下离心5分钟,在室温下收集细胞5分钟
  26. 用200μl显影介质洗涤细胞,并在室温下以365×g离心5分钟。
  27. 将沉淀重悬在200μl的显影介质中,并在室温下在黑暗中孵育细胞16小时
  28. 在两个空孔中,加入一滴每种BD补偿珠小瓶(阴性对照和抗小鼠IgK)。
  29. 通过在4℃下以365×g离心5分钟收集细胞和珠子
  30. 通过加入200μlFACS染色缓冲液洗涤细胞和珠一次
  31. 通过在4℃下以365×g离心5分钟收集细胞,并将沉淀重悬于100μl含有1/100稀释的抗CD3-APC的免疫染色溶液(在FACS染色缓冲液中) -Cy7和抗-CD4-PE-Cy7的1/50稀释液。 不要染色2补偿控制和珠。 用含有1/100稀释的抗CD3-APC或1/50稀释的抗CD4-PE-Cy7的FACS染色缓冲液染色CompBeads。
  32. 在4℃孵育30分钟。
  33. 通过在4℃下以365×g离心5分钟收集细胞和珠子
  34. 用200μlFACS染色缓冲液洗涤细胞和珠子
  35. 将细胞在补充有1.2%多聚甲醛的FACS染色缓冲液中在4℃固定24小时
  36. 在FACS仪器上获取样品。该组样品应包括:未加载的未染色细胞,加载CCF2的未染色细胞,CD3-APC-Cy7染色珠,CD4-PE-Cy7染色珠,负载CCF2的未感染对照和免疫染色,加载CCF2和免疫染色。除了测定的参数(未裂解和切割的底物,APC-Cy7和PE-Cy7)之外,确保获得FITC通道中的发射。电压应使用未感染的,免疫染色的样品设置。
  37. 将fcs文件导入FlowJo软件。使用对应于CompBeads的fcs文件来补偿APC-Cy7和PE-Cy7,使用CCF2加载和卸载样本在FITC通道中进行补偿。对所有样品应用补偿。
  38. 如图2所示对单元格进行门控。

代表数据



图1.融合检测原理


图2.代表性数据 A.测量HIV-1融合到静息PBL培养物的门控策略。细胞群首先在前向散射区(FSC-A)和侧向散射区(SSC-A)上门控。然后在具有FSC-A和FSC-H的FACS图上门控单细胞。 CD4T细胞群在APC-Cy7和PE-Cy7上门控。在表示裂解的CCF2对未裂解的CCF2的FACS图中,在未感染的样品上绘制融合门。 B.对于未感染的样品(NI),X4-趋向性NL4-3,R5-趋向性81A和亚克隆到TN6-GFP中的初级HIV-1包膜(Env)获得的代表性融合FACS图(Cavrois等al。,2011; Cavrois ,,2014)。

笔记

  1. 尽管在对HIV-1融合的易感性方面存在供体 - 供体变异性,但通过81A的正常化显示了给定初级Env介导融合的相对能力的非常好的评估(Cavrois等人, 2014)。

食谱

  1. HBSS
    280 mM NaCl 10 mM KCl
    1.5mM Na 2 HPO 4
    12mM葡萄糖 (2-羟乙基哌嗪) - N' - (2-乙磺酸)(HEPES)(pH 7.05)的缓冲液 储存于-20°C
  2. 荧光激活细胞分选(FACS)染色缓冲液
    1×磷酸盐缓冲盐水(PBS)
    2%胎牛血清 储存在4°C
  3. Dulbecco改良的Eagle培养基(DMEM)培养基 DMEM
    10%胎牛血清 100 U/ml青霉素
    100μg/ml链霉素
  4. RPMI培养基
    RPMI 1640
    10%胎牛血清 100 U/ml青霉素
    100μg/ml链霉素
  5. CCF2加载溶液
    1. 将CCF2-AM重悬于二甲基亚砜(CCF2试剂盒的溶液A)中以产生储备溶液(1mM CCF2-AM)。 分成等份,在-80℃下在黑暗中储存
    2. 通过涡旋混合1μl1mM CCF2-AM与9μl含有100mg/ml Pluronic-F127和0.1%乙酸的溶液(CCF2试剂盒的溶液B)混合。
    3. 加入1ml CO 2独立培养基并再次涡旋
  6. 在250mM NaOH中制备的丙磺舒储备溶液(250mM) 分成等份,储存在-20°C
  7. 开发媒体
    2.5mM丙磺舒
    10%胎牛血清的CO 2非依赖性培养基中

致谢

该测定首次公开于(Cavrois等人,2002)和在(Cavrois等人,2004)中详细描述的方案。 融合测定还允许对由其他病毒(包括埃博拉病毒(Yonezawa等人,2005)和henipavirus(Wolf等人,2009))介导的融合进行研究。 我们要感谢Tsien教授的团队领导开发CCF2,BlaM底物(Zlokarnik等人,1998)。

参考文献

  1. Cavrois,M.,De Noronha,C.and Greene,W.C。(2002)。 在原代T淋巴细胞中检测HIV-1病毒体融合的敏感和特异性的酶基测定。 a> Nat Biotechnol 20(11):1151-1154
  2. Cavrois,M.,Neidleman,J.,Bigos,M。和Greene,W.C。(2004)。 基于荧光共振能量转移的HIV-1病毒颗粒融合测定法 方法Mol Biol 263:333-344
  3. Cavrois,M.,Neidleman,J.,Galloway,N.,Derdeyn,C.A.,Hunter,E.and Greene,W.C。 测量来自原发性病毒分离株的信封介导的HIV融合。 53(1):34-38。
  4. Cavrois,M.,Neidleman,J.,Santiago,M.L.,Derdeyn,C.A.,Hunter,E.and Greene,W.C。(2014)。 增强融合和病毒粒子结合用于具有紧凑V1/V2结构域的HIV-1亚型C包膜糖蛋白。/a> J Virol 88(4):2083-2094。
  5. Hayden,M.S.,Palacios,E.H。和Grant,R.M。(2003)。 使用荧光连锁抗原定量测定法实时定量HIV-1 p24和SIV p27。/a> AIDS 17(4):629-631。
  6. Maiti,M.,Grant,R。和Cavrois,M.(2014)。 用于HIV-1 p24Gag的快速定量的荧光连锁抗原定量(FLAQ)测定法 <生物协议 4(24):e1366
  7. Cavrois,M.,Neidleman,J.,Bigos,M。和Greene,W.C。(2004)。 基于荧光共振能量转移的HIV-1病毒颗粒融合测定法 方法Mol Biol 263:333-344
  8. Cavrois,M.,Neidleman,J.,Galloway,N.,Derdeyn,C.A.,Hunter,E.and Greene,W.C。 测量来自原发性病毒分离株的信封介导的HIV融合。 53(1):34-38。
  9. Cavrois,M.,Neidleman,J.,Santiago,M.L.,Derdeyn,C.A.,Hunter,E.and Greene,W.C。(2014)。 增强融合和病毒粒子结合用于具有紧凑V1/V2结构域的HIV-1亚型C包膜糖蛋白。/a> J Virol 88(4):2083-2094。
  10. Hayden,M.S.,Palacios,E.H。和Grant,R.M。(2003)。 使用荧光连锁抗原定量测定法实时定量HIV-1 p24和SIV p27。/a> AIDS 17(4):629-631。
  11. Maiti,M.,Grant,R。和Cavrois,M.(2014)。 用于HIV-1 p24Gag的快速定量的荧光连锁抗原定量(FLAQ)测定法 <生物协议 4(24):e1366...
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引用:Cavrois, M., Neidleman, J. and Greene, W. C. (2014). HIV-1 Fusion Assay . Bio-protocol 4(16): e1212. DOI: 10.21769/BioProtoc.1212.
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