搜索

Affinofile Assay for Identifying Macrophage-Tropic HIV-1
Affinofile 分析法鉴定巨噬细胞热带性HIV-1   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The ability to enter monocyte-derived macrophage (MDM) in vitro is commonly used to define macrophage-tropic HIV-1 despite the fact that viruses vary continuously in their ability to enter MDMs in vitro, and MDMs vary in their ability to support HIV-1 entry (Joseph et al., 2014; Peters et al., 2006). This makes it difficult to distinguish viruses that are adapted to replicating in macrophage from those that are adapted to replicating in T cells. We use the Affinofile cell line ( Johnston et al., 2009) to assay for macrophage tropism by capitalizing on the fact that macrophage-tropic HIV-1 has an enhanced ability to enter cells expressing low levels of CD4 (Joseph et al., 2014; Peters et al., 2006; Duenas-Decamp et al., 2009; Dunfee et al., 2006; Gorry et al., 2002; Martin-Garcia et al., 2006; Peters et al., 2004) and Affinofile cells can be induced to express a wide range of CD4 densities (Johnston et al., 2009). We induce Affinofile cells to express either high or low CD4, infect those cells with pseudotyped reporter virus, and quantify percent infectivity at low CD4 relative to infectivity at high CD4. Macrophage-tropic viruses have an enhanced ability to infect at low CD4. Using this approach we have found that macrophage-tropic strains of HIV-1 are relatively rare and that most HIV-1 variants require high levels of CD4 to enter cells, a phenotype we have referred to as R5 T cell-tropic.

Materials and Reagents

  1. Affinofile cells (Johnston et al., 2009)
  2. Luciferase Assay System (Promega Corporation, catalog number: E1501 )
  3. 5x Reporter Lysis Buffer (Promega Corporation, catalog number: E397A )
  4. PE-conjugated anti-CD4 antibody (clone RPA-T4) (BD, catalog number: 555347 )
  5. PE-conjugated anti-CCR5 antibody (clone 2D7) (BD, catalog number: 555993 )
  6. Aqua live/dead stain (Life Technologies, catalog number: L34957 )
  7. Env-pseudotyped luciferase reporter virus stock (frozen and tittered)
  8. Blasticidin (Life Technologies, catalog number: A11139-03 )
  9. FBS (Atlanta Biologicals, catalog number: S12850 )
  10. DMEM-H (Cellgro, catalog number: 10-013-CV )
  11. 10% Buffered Formalin (Thermo Fisher Scientific, catalog number: SF100 )
  12. Poly-L-Lysine (Sigma-Aldrich, catalog number: P4707 ) (see Recipes)
  13. Ponasterone A (Life Technologies, catalog number: 45-0478 ) (see Recipes)
  14. Doxycycline (Sigma-Aldrich, catalog number: D9891 ) (see Recipes)
  15. DMEM-F10/B (see Recipes)
  16. 1x staining solution (see Recipes)
  17. Fixing solution (see Recipes)

Equipment

  1. 96-well plate (sterile, black) (Corning, Costar®, catalog number: 3916 )
  2. 24 well plate
  3. 37 °C, 5% CO2 cell culture incubator (BSL-2)
  4. Hemocytometer
  5. Light microscope
  6. PE-conjugated QuantiBRITE beads (BD, catalog number: 340495 )
  7. Incubated plate centrifuge (BSL-2)
  8. Laminar flow biosafety cabinet (BSL-2)
  9. Plate-reading luminometer

Procedure

  1. For high versus low CD4 comparison
    Day 1
    1. Add 50 µl of diluted Poly-L-Lysine to sterile, black 96 well plates and incubate at 37 °C for 20 min.  Remove Poly-L-Lysine.
    2. Treat Affinofile cells with trypsin-EDTA and harvest in DMEM-F10/B.
    3. Count cells and dilute to a concentration of 1.8 x 105 cells/ml in DMEM-F10/B.
    4. Plate 100 µl of cells/well in 96 well plates.
    5. Plate 0.75 ml of cells/well in 24 well plate for flow cytometry. Each well will be stained for flow cytometry. Plate enough cells to perform the necessary isotype and compensation controls.
    6. Incubate cells at 37 °C overnight.
    Day 2
    1. Prepare media for high and low CD4 induction.
      1. Dilute Doxycycline (Doxy) stock to 4x in DMEM-F10/B. A concentration of 24 ng/ml corresponds to 4x the concentration necessary for maximum CD4 induction.
      2. Dilute Ponasterone A (Pon A) stock to 4x in DMEM-F10/B. A concentration of 20 µM corresponds to 4x the concentration necessary for maximum CCR5 induction.
      3. Low CD4 induction medium (Low CD4/High CCR5): Mix equal volumes of 4x Pon A and DMEM-F10/B (=2x Pon A and 0x Doxy).
      4. High CD4 induction medium (High CD4/High CCR5): Mix equal volumes of 4x Pon A and 4x Doxy (=2x Pon A and 2x Doxy).
    2. Induce cells for infection.
      1. Remove 50 µl of medium from each well of the 96-well plate.
      2. For each virus being tested, add 50 µl of low CD4 induction medium to 3 wells (=1x Pon A and 0x Doxy) and 50 µl of High CD4 induction medium to 3 wells (=1x Pon A and 1x Doxy).
      3. Incubate cells at 37 °C.
    3. Induce cells for flow cytometry.
      1. Remove all medium from the 24 well plate, replace with 750 µl of warm DMEM-F10/B and 250 µl of either 4x Doxy or 4x Pon A (=1x desired amount of Pon A or Doxy).  
      2. Incubate cells at 37 °C.
    Day 3
    1. Infect cells 18-24 h after adding drugs (Pon A and Doxy).
      1. Remove induction medium from wells and immediately replace with 50 µl of warm DMEM-D10/B.
      2. Warm plate centrifuge to 37 °C.
      3. Thaw pseudovirus stock and dilute in DMEM-D10/B so that 50 µl of diluted virus produces a desired signal when titered on Affinofile cells expressing maximum CD4 and CCR5. This is determined by pre-titering the virus to ensure the amount of virus added is within the linear dose-response curve for the readout (in this case luciferase) and many fold above the assay background. As an additional control, include a dilution curve of a high titer virus to ensure all signals are within the linear range.
      4. Add 50 µl of diluted virus to each well of a 96-well plate (3 High CD4 wells and 3 Low CD4 wells per virus).
      5. Centrifuge plates at 2,000 rpm (849 x g) for 2 h at 37 °C (spinoculation; O'Doherty et al., 2000).
      6. Incubate cells at 37 °C for 48 h.
    2. Prepare cells for flow cytometry.
      1. Remove medium from a well of a 24 well plate, then use 1 ml of cold PBS to harvest the cells from that well and store the cells in eppendorf tubes on ice until all wells are harvested.
      2. Centrifuge the harvested cells in a table top microcentrifuge at 4 °C at 2,600 rpm for 10 min.  
      3. While centrifuging, make staining solutions (Recipe 5).    
      4. Resuspend the cells in 50 µl of staining solution and stain at room temperature, in the dark, for 60 min.
      5. Add, with gentle mixing, 1 ml of fresh fixing solution (Recipe 6).
      6. Centrifuge in cold room at 2,600 rpm for 10 min, dispose of supernatant.
      7. Resuspend cells in 1 ml PBS.
      8. Centrifuge in cold room at 2,600 rpm for 10 min.  
      9. Resuspend cells in 300 µl PBS.
      10. Store in the dark at 4 °C until ready to analyze.
      11. Analyze cells and QuantiBRITE beads using flow cytometer equipped with a 405 nm laser and a filter that measures emissions at approximately 526 nm and a 488 nm laser with a filter that measures emissions at approximately 578 nm.
      12. Use Quantibrite beads, with their four known levels of PE, to determine the relationship between mean fluorescence and number of PE molecules (Figure 1). This relationship can be used to translate the PE fluorescence of stained cells into mean number of CD4 or CCR5 antibody binding sites (ABS) per cell. This method requires that cells be stained with saturating levels of antibody and assumes that each antibody is conjugated to a single PE molecule.


        Figure 1. QuantiBRITE beads are used to quantify the relationship between PE fluorescence and number of PE molecules attached to a bead/cell. A) QuantiBRITE beads are analyzed by flow cytometry to calculate the mean, log PE fluorescence of the four bead populations, each conjugated to a different, known number of PE molecules. B) The relationship between log (PE molecules / bead) and log (log PE) is then determined. This relationship can then be used to translate the mean PE fluorescence of stained cells into the mean number of CD4 or CCR5 antibody binding sites (ABS) per cell.

    Day 5
    1. Harvest infected cells 48 h after infection.
      1. Remove medium from wells.
      2. Gently rinse 2x with warm PBS, discard PBS after each wash.
      3. Add 50 µl of 1x Reporter Lysis Buffer and incubate at room temperature for 15 min.
      4. Seal plates with aluminum foil covers and freeze at -80 °C for at least 2 h.
      5. Thaw plates and Luciferase Assay System reagent at room temperature.
      6. Prepare Luciferase Assay reagents following the manufacturer’s protocol, and read plate using a plate-reading luminometer.
      7. Calculate percent infectivity at low CD4 relative to infectivity at high CD4 for each individual virus.

  2. For continuous CD4 comparison
    Macrophage-tropism can also be assessed by infecting Affinofile cells expressing a wide range of CD4 densities (Joseph et al., 2014). CD4 usage curves reveal that, relative to T cell-tropic viruses, macrophage-tropic viruses are significantly better at infecting cells expressing low levels of CD4, reach 50% infectivity at lower CD4 levels (CD4 ED50) and often approach a plateau level of infectivity at a lower CD4 density (Figure 2; Joseph et al., 2014).  This procedure is identical to the or ‘Procedure for High Versus Low CD4 Comparison’ (part A above)  except that cells are induced to express 10 levels of CD4 and 30 wells of a 96-well plate are needed to test each virus (10 CD4 levels x 3 replicates per level).


    Figure 2. Dose-response curves for infectivity of a macrophage-tropic virus (Ba-L) and a T cell-tropic viruses (JRCSF) to Affinofile cells expressing various densities of CD4. The sensitivity of pseudotyped viruses to CD4 levels was assessed by measuring their ability to infect Affinofile cells expressing 10 CD4 levels (1,425, 4,590, 4,981, 9,374, 22,667, 33,842, 46,204, 58,153, 69,897, and 81,649 CD4 ABS per cell), with CCR5 fully induced. In this assay, the vast majority of blood-derived viruses have an infectivity profile similar to that of JRCSF ( Ping et al., 2013).

    Day 2
    1. Prepare media for continuous CD4 comparison.
      1. Dilute Doxycycline stock to 24 ng/ml in DMEM-F10/B.
      2. Serially dilute Doxycycline to the following concentrations:  6, 4, 2.8, 2, 1.2, 0.8, 0.4, 0.28 ng/ml.
      3. Dilute Ponasterone A stock to 20 µM in DMEM-F10/B.
      4. Induction medium (10 levels of Doxycycline and 1 high level of Pon A): Mix equal volumes of 4x Doxycycline and 4x Ponasterone A (Table 1).

        Table 1. Dilutions of Doxycycline and Ponasterone A to induce ten levels of CD4 expression and a single, high level of CCR5 expression
        4x Doxycycline (ng/ml)    
        4x Ponasterone A (µM)    
        1x Final Doxycycline concentration    
        1x Final Ponasterone A concentration
        24   
        20  
        6   
        5
        6   
        20   
        1.5   
        5
        4  
        20   
        1   
        5
        2.8   
        20   
        0.7   
        5
        2   
        20   
        0.5   
        5
        1.2   
        20   
        0.3   
        5
        0.8   
        20   
        0.2   
        5
        0.4   
        20   
        0.1   
        5
        0.28   
        20   
        0.07   
        5
        0   
        20   
        0  
        5

    2. Induce cells for infection.
      1. Remove 50 µl of media from each well of the 96-well plate.
      2. For each virus being tested, at each Doxycycline level, add 50 µl of each induction medium to 3 wells (10 induction levels x 3 replicates = 30 wells / virus).
      3. Incubate cells at 37 °C.
    3. Induce cells for flow cytometry
      1. Remove all medium from the 24 well plate, replace with 750 µl of warm DMEM-F10/B and 250 µl of either 4x Doxy or 4x Pon A (=1x desired amount of Pon A or Doxy).  
      2. Incubate cells at 37 °C.
        Days 3 and 5 are the same as described in ‘Procedure for High Versus Low CD4 Comparison’ (part A above).

Recipes

  1. Poly-L-Lysine (0.1 mg/ml)
    Diluted 10-1 in sterile PBS (10 µg/ml) and stored at 4 °C
  2. Ponasterone A
    Diluted in 50% ethanol to 1 mM and stored in 100 µl aliquots at -20 °C
  3. Doxycycline
    Diluted in sterile water to 10 mg/ml and frozen in 20 µl aliquots at -20 °C (do not freeze-thaw)
  4. DMEM-F10/B
    450 ml Cellgro DMEM-H
    2.5 ml of blasticidin (final conc = 50 mg/ml, do not freeze/thaw blasticidin)
    50 ml of dialyzed FBS (final = 10%)
    Stored at 4 °C
  5. 1x staining solution
    3 µl of antibody (anti-CD4, anti-CCR5 or isotype controls)
    2 µl of aqua live/dead stain
    45 µl of PBS
    1% FBS
  6. Fixing solution
    200 µl of 10% formalin
    800 µl H2O

Acknowledgement

We would like to thank Kathryn T. Arrildt for helpful comments that improved this protocol. This work was funded by the National Institutes of Health grant R37 AI44667 to RS.

References

  1. Dunfee, R. L., Thomas, E. R., Gorry, P. R., Wang, J., Taylor, J., Kunstman, K., Wolinsky, S. M. and Gabuzda, D. (2006). The HIV Env variant N283 enhances macrophage tropism and is associated with brain infection and dementia. Proc Natl Acad Sci U S A 103(41): 15160-15165.
  2. Duenas-Decamp, M. J., Peters, P. J., Burton, D. and Clapham, P. R. (2009). Determinants flanking the CD4 binding loop modulate macrophage tropism of human immunodeficiency virus type 1 R5 envelopes. J Virol 83(6): 2575-2583.
  3. Gorry, P. R., Taylor, J., Holm, G. H., Mehle, A., Morgan, T., Cayabyab, M., Farzan, M., Wang, H., Bell, J. E., Kunstman, K., Moore, J. P., Wolinsky, S. M. and Gabuzda, D. (2002). Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate. J Virol 76(12): 6277-6292.
  4. Johnston, S. H., Lobritz, M. A., Nguyen, S., Lassen, K., Delair, S., Posta, F., Bryson, Y. J., Arts, E. J., Chou, T. and Lee, B. (2009). A quantitative affinity-profiling system that reveals distinct CD4/CCR5 usage patterns among human immunodeficiency virus type 1 and simian immunodeficiency virus strains. J Virol 83(21): 11016-11026.
  5. *Joseph, S. B., Arrildt, K. T., Swanstrom, A. E., Schnell, G., Lee, B., Hoxie, J. A. and Swanstrom, R. (2014). Quantification of Entry Phenotypes of Macrophage-Tropic HIV-1 across a Wide Range of CD4 Densities. J Virol 88(4): 1858-1869.
  6. O'Doherty, U., Swiggard, W. J. and Malim, M. H. (2000). Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol 74(21): 10074-10080.
  7. Peters, P. J., Bhattacharya, J., Hibbitts, S., Dittmar, M. T., Simmons, G., Bell, J., Simmonds, P. and Clapham, P. R. (2004). Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages. J Virol 78(13): 6915-6926.
  8. Peters, P. J., Sullivan, W. M., Duenas-Decamp, M. J., Bhattacharya, J., Ankghuambom, C., Brown, R., Luzuriaga, K., Bell, J., Simmonds, P., Ball, J. and Clapham, P. R. (2006). Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis. J Virol 80(13): 6324-6332.
  9. Ping, L. H., Joseph, S. B., Anderson, J. A., Abrahams, M. R., Salazar-Gonzalez, J. F., Kincer, L. P., Treurnicht, F. K., Arney, L., Ojeda, S., Zhang, M., Keys, J., Potter, E. L., Chu, H., Moore, P., Salazar, M. G., Iyer, S., Jabara, C., Kirchherr, J., Mapanje, C., Ngandu, N., Seoighe, C., Hoffman, I., Gao, F., Tang, Y., Labranche, C., Lee, B., Saville, A., Vermeulen, M., Fiscus, S., Morris, L., Karim, S. A., Haynes, B. F., Shaw, G. M., Korber, B. T., Hahn, B. H., Cohen, M. S., Montefiori, D., Williamson, C., Swanstrom, R., Study, C. A. I. and the Center for, H. I. V. A. V. I. C. (2013). Comparison of viral Env proteins from acute and chronic infections with subtype C human immunodeficiency virus type 1 identifies differences in glycosylation and CCR5 utilization and suggests a new strategy for immunogen design. J Virol 87(13): 7218-7233.
  10. Martin-Garcia, J., Cao, W., Varela-Rohena, A., Plassmeyer, M. L. and Gonzalez-Scarano, F. (2006). HIV-1 tropism for the central nervous system: Brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor. Virology 346(1): 169-179.

简介

体外进入单核细胞衍生的巨噬细胞(MDM)的能力通常用于定义巨噬细胞向性HIV-1,尽管事实上病毒在体外进入MDM的能力不断变化。/em>,并且MDMs支持HIV-1进入的能力不同(Joseph等人,2014; Peters等人,2006)。这使得难以将适于在巨噬细胞中复制的病毒与适于在T细胞中复制的病毒区分开。我们使用Affinofile细胞系(Johnston等人,2009)通过利用巨噬细胞嗜性HIV-1具有增强的进入表达低水平CD4的细胞的能力的事实来测定巨噬细胞趋向性(Joseph等人,2014; Peters等人,2006; Duenas-Decamp等人,2009; Dunfee等人et al。,2006; Gorry et al。,2002; Martin-Garcia et al。,2006; Peters et al。, ,2004),并且亲和素细胞可以被诱导以表达宽范围的CD4密度(Johnston等人,2009)。我们诱导Affinofile细胞表达高或低CD4,用假型报告病毒感染那些细胞,并且量化在低CD4相对于高CD4的感染性的百分比感染性。巨噬细胞嗜性病毒在低CD4下具有增强的感染能力。使用这种方法,我们已经发现HIV-1的巨噬细胞向性菌株相对稀少,并且大多数HIV-1变体需要高水平的CD4进入细胞,我们称为R5 T细胞向性的表型。

材料和试剂

  1. 亲和体细胞(Johnston等人,2009)
  2. 萤光素酶测定系统(Promega Corporation,目录号:E1501)
  3. 5x报道裂解缓冲液(Promega Corporation,目录号:E397A)
  4. PE结合的抗CD4抗体(克隆RPA-T4)(BD,目录号:555347)
  5. PE结合的抗CCR5抗体(克隆2D7)(BD,目录号:555993)
  6. Aqua活/死染色(Life Technologies,目录号:L34957)
  7. Env-假型荧光素酶报道病毒原种(冷冻和滴定)
  8. 杀稻瘟素(Life Technologies,目录号:A11139-03)
  9. FBS(Atlanta Biologicals,目录号:S12850)
  10. DMEM-H(Cellgro,目录号:10-013-CV)
  11. 10%缓冲福尔马林(Thermo Fisher Scientific,目录号:SF100)
  12. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)(参见Recipes)
  13. Ponasterone A(Life Technologies,目录号:45-0478)(参见配方)
  14. 多西环素(Sigma-Aldrich,目录号:D9891)(参见Recipes)
  15. DMEM-F10/B(参见配方)
  16. 1x染色溶液(参见配方)
  17. 固定解决方案(参见配方)

设备

  1. 96孔板(无菌,黑色)(Corning,Costar ,目录号:3916)
  2. 24孔板
  3. 37℃,5%CO 2细胞培养箱(BSL-2)
  4. 血细胞计数器
  5. 光学显微镜
  6. PE偶联的QuantiBRITE珠(BD,目录号:340495)
  7. 孵育板离心机(BSL-2)
  8. 层流生物安全柜(BSL-2)
  9. 读板光度计

程序

  1. 对于高与低CD4比较
    第1天
    1. 加入50μl稀释的聚-L-赖氨酸到无菌,黑色96孔板中,并在37℃孵育20分钟。 去除聚-L-赖氨酸。
    2. 用胰蛋白酶-EDTA处理Affinofile细胞并在DMEM-F10/B中收获。
    3. 计数细胞并在DMEM-F10/B中稀释至浓度为1.8×10 5个细胞/ml。
    4. 板100微升细胞/井在96孔板。
    5. 在24孔板中铺平板0.75ml细胞/孔用于流式细胞术。 每 孔将被流式细胞术染色。 板足够的单元格执行 必要的同种型和补偿控制
    6. 孵育细胞在37℃过夜。
    第2天
    1. 准备培养基用于高和低CD4诱导。
      1. 稀 多西环素(Doxy)原液在DMEM-F10/B中为4倍。 浓度为24 ng/ml对应于最大CD4所需的浓度的4倍 感应
      2. 稀释Ponasterone A(Pon A)股票到4x英寸 DMEM-F10/B。 20μM的浓度对应于4倍的浓度   为最大CCR5诱导所必需
      3. 低CD4诱导培养基(低CD4 /高CCR5):混合等体积的4x Pon A和DMEM-F10/B(= 2x Pon A和Ox Doxy)。
      4. 高CD4诱导培养基(高CD4 /高CCR5):混合等体积的4x Pon A和4x Doxy(= 2x Pon A和2x Doxy)。
    2. 诱导细胞感染。
      1. 从96孔板的每个孔中取出50μl培养基
      2. 对于每个被测试的病毒,加入50μl低CD4诱导培养基至3   孔(= 1×Pon A和0×Doxy)和50μl高CD4诱导培养基   3个孔(= 1×Pon A和1×Doxy)
      3. 在37℃下孵育细胞。
    3. 诱导细胞流式细胞术。
      1. 从24孔板中取出所有培养基,更换为750μl温热 DMEM-F10/B和250μl的4x Doxy或4x Pon A(=所需量的1x 的Pon A或Doxy)。  
      2. 在37℃下孵育细胞。
    第3天
    1. 在加入药物后18-24小时感染细胞(Pon A和Doxy)。
      1. 从孔中取出感应培养基,立即更换为50微升温暖的DMEM-D10/B
      2. 将板温箱离心至37℃
      3. 解冻假病毒储液和稀释在DMEM-D10/B,使50微升 当在Affinofile细胞上滴定时,稀释的病毒产生期望的信号   表达最大的CD4和CCR5。 这是通过预滴定确定的   病毒以确保病毒添加量在线性范围内 读出的剂量 - 反应曲线(在这种情况下是荧光素酶)和许多 高于测定背景。 作为附加控制,包括a 高滴度病毒的稀释曲线,以确保所有信号都在内 线性范围
      4. 向96孔板(每个病毒3个高CD4孔和3个低CD4孔)的每个孔中加入50μl稀释的病毒。
      5. 在2,000rpm(849×g)下在37℃离心板2小时(spinoculation; O'Doherty等人,2000)。
      6. 孵育细胞在37℃下48小时。
    2. 准备细胞流式细胞术。
      1. 从24孔板的孔中取出培养基,然后使用1毫升冷 PBS从该孔收获细胞并将细胞储存在eppendorf中 管子在冰上,直到收获所有的孔
      2. 将收获的细胞在台式微量离心机中在4℃下以2,600rpm离心10分钟。  
      3. 在离心的同时,制作染色溶液(配方5)。  
      4. 将细胞重悬于50μl染色溶液中,并在室温下,在黑暗中染色60分钟
      5. 在温和混合下加入1ml新鲜的定影溶液(配方6)
      6. 在冷室中以2,600rpm离心10分钟,处理上清液。
      7. 重悬细胞在1ml PBS。
      8. 在冷室中以2,600rpm离心10分钟。  
      9. 重悬细胞在300微升PBS
      10. 储存在黑暗中4°C,直到准备分析
      11. 使用配有的流式细胞仪分析细胞和QuantiBRITE珠   405nm激光器和大约测量发射的滤波器 526 nm和488 nm激光器,带有测量处的发射的滤波器 约578nm。
      12. 使用Quantibrite珠,与他们的四 已知水平的PE,以确定平均值之间的关系 荧光和PE分子的数量(图1)。这种关系 可用于将染色细胞的PE荧光翻译成平均值 每个细胞的CD4或CCR5抗体结合位点(ABS)的数目。这个方法  需要细胞用饱和水平的抗体染色 假设每个抗体与单个PE分子缀合

        图  QuantiBRITE珠用于定量PE之间的关系 荧光和连接到珠/细胞的PE分子的数目。 A) 通过流式细胞术分析QuantiBRITE珠以计算平均值, log PE荧光的四个珠子群体,每个缀合到a 不同的,已知数量的PE分子。 B)日志之间的关系  (PE分子/珠)和log(log PE)。这个 关系可以用于平均PE荧光的平移 将细胞染色成CD4或CCR5抗体结合位点的平均数  (ABS)。

    第5天
    1. 收获感染后48小时感染的细胞。
      1. 从井中取出培养基。
      2. 用温热的PBS轻轻冲洗2次,每次冲洗后丢弃PBS
      3. 加入50μl1x Reporter Lysis Buffer,室温孵育15分钟
      4. 带有铝箔盖的密封板,并在-80℃下冷冻至少2小时
      5. 解冻板和萤光素酶测定系统试剂在室温
      6. 根据制造商的方案制备荧光素酶测定试剂,并使用读板光度计读取平板
      7. 计算在低CD4下的感染性百分比相对于每种病毒的高CD4的感染性

  2. 用于连续CD4比较
    巨噬细胞向性也可以通过感染表达大范围CD4密度的亲和素细胞来评估(Joseph等人,2014)。 CD4使用曲线显示,相对于T细胞嗜性病毒,巨噬细胞嗜性病毒在感染表达低水平CD4的细胞方面显着更好,在较低CD4水平(CD4 ED50)达到50%感染性,并且经常接近平稳水平的感染性 在低CD4密度(图2; Joseph等人, 2014)。该程序与"高或低CD4比较的程序"(上文A部分)相同,除了细胞被诱导表达10个水平的CD4和96孔板的30个孔需要测试每个病毒(10个CD4水平×每级3个重复)。


    图2.巨噬细胞向性病毒(Ba-L)和T细胞嗜性病毒(JRCSF)对表达各种密度的CD4的Affinofile细胞的感染性的剂量 - 反应曲线。假型通过测量其感染表达10种CD4水平(1,425,4,590,4,981,9,374,22,667,33,842,46,204,58,153,69,897,和81,649个CD4 ABS /细胞)的亲和素细胞的能力,评估病毒与CD4水平的比例,其中CCR5完全诱导。在该测定中,绝大多数血源性病毒具有与JRCSF相似的感染性概况(Ping等人,2013)。

    第2天
    1. 准备培养基进行连续CD4比较。
      1. 在DMEM-F10/B中将多西环素原液稀释至24ng/ml。
      2. 将多西环素连续稀释至以下浓度: 6,4,2.8,2,1.2,0.8,0.4,0.28ng/ml。
      3. 稀释Ponasterone A股票在DMEM-F10/B的20μM。
      4. 诱导培养基(10个水平的多西环素和1个高水平的Pon A):   混合等体积的4x多西环素和4x Ponasterone A(表1)
        表   多西环素和松甾酮A的稀释液诱导十个水平的 CD4表达和单一,高水平的CCR5表达
        4x 多西环素(ng/ml)   
        4x Ponasterone A(μM)   
        1x最终多西环素 浓度   
        1x最终Ponasterone浓度
        24   
        20  
        6   
        5
        6   
        20   
        1.5   
        5
        4  
        20   
        1   
        5
        2.8   
        20   
        0.7   
        5
        2   
        20   
        0.5   
        5
        1.2   
        20   
        0.3   
        5
        0.8   
        20   
        0.2   
        5
        0.4   
        20   
        0.1   
        5
        0.28   
        20   
        0.07   
        5
        0   
        20   
        0  
        5

    2. 诱导细胞感染。
      1. 从96孔板的每个孔中取出50μl培养基。
      2. 对于每个被测试的病毒,在每个多西环素水平,添加50μl 每个诱导培养基至3个孔(10个诱导水平×3个重复= 30孔/病毒)。
      3. 在37℃下孵育细胞。
    3. 诱导细胞流式细胞术
      1. 从24孔板中取出所有培养基,更换为750μl温热 DMEM-F10/B和250μl的4x Doxy或4x Pon A(=所需量的1x 的Pon A或Doxy)。  
      2. 在37℃下孵育细胞。
        第3天和第5天与"用于高对比CD4低比较的程序"(上文A部分)中所述的相同。

食谱

  1. 聚-L-赖氨酸(0.1mg/ml)
    在无菌PBS(10μg/ml)中稀释10μL-1并在4℃下贮存
  2. Ponasterone A
    在50%乙醇中稀释至1mM,并以100μl等分试样储存在-20℃下
  3. 多西环素
    在无菌水中稀释至10 mg/ml,并在-20°C下冷冻成20μl等分试样(不冻融)
  4. DMEM-F10/B
    450ml Cellgro DMEM-H
    2.5ml杀稻瘟素(最终浓度= 50mg/ml,不冻/杀灭杀稻瘟素) 50ml透析的FBS(最终= 10%) 储存在4°C
  5. 1×染色溶液
    3μl抗体(抗CD4,抗CCR5或同种型对照)
    2μl水生活/死亡污迹
    45微升PBS
    1%FBS
  6. 固定解决方案
    200μl10%福尔马林
    800μlH 2 O x/s

确认

我们要感谢Kathryn T. Arrildt的有益意见,改进了这个协议。这项工作由国家卫生研究院资助R37 AI44667到RS。

参考文献

  1. Dunfee,R.L.,Thomas,E.R.,Gorry,P.R.,Wang,J.,Taylor,J.,Kunstman,K.,Wolinsky,S.M.and Gabuzda,D.(2006)。 HIV Env变体N283增强巨噬细胞嗜性,并与脑感染和痴呆相关。 Proc Natl Acad Sci USA 103(41):15160-15165。
  2. Duenas-Decamp,M.J.,Peters,P.J.,Burton,D。和Clapham,P.R。(2009)。 侧翼于CD4结合环的决定簇调节人类免疫缺陷病毒1型R5包膜的巨噬细胞嗜性。 J Virol 83(6):2575-2583。
  3. Gorry,PR,Taylor,J.,Holm,GH,Mehle,A.,Morgan,T.,Cayabyab,M.,Farzan,M.,Wang,H.,Bell,JE,Kunstman,K.,Moore,JP ,Wolinsky,SM和Gabuzda,D。(2002)。 提高CCR5亲和力,降低神经毒性原发性人类免疫缺陷病毒1型分离株的CCR5/CD4依赖性。/a> J Virol 76(12):6277-6292。
  4. Johnston,S.H.,Lobritz,M.A.,Nguyen,S.,Lassen,K.,Delair,S.,Posta,F.,Bryson,Y.J.,Arts,E.J.,Chou,T.and Lee, 一种定量亲和力分析系统,可揭示人类免疫缺陷病毒1型之间的不同CD4/CCR5使用模式,猴免疫缺陷病毒毒株。 J Virol 83(21):11016-11026。
  5. * Joseph,S.B.,Arrildt,K.T.,Swanstrom,A.E.,Schnell,G.,Lee,B.,Hoxie,J.A。和Swanstrom,R。(2014)。 巨噬细胞HIV-1在广泛CD4密度下的表型的定量。 a> J Virol 88(4):1858-1869。
  6. O'Doherty,U.,Swiggard,W.J.and Malim,M.H。(2000)。 人类免疫缺陷病毒1型spinoculation通过病毒结合增强感染。 J Virol 74(21):10074-10080。
  7. Peters,P.J.,Bhattacharya,J.,Hibbitts,S.,Dittmar,M.T.,Simmons,G.,Bell,J.,Simmonds,P.and Clapham,P.R。(2004)。 从艾滋病患者的脑和淋巴结组织扩增的1型人类免疫缺陷病毒R5包膜的生物学分析神经病理学揭示两种截然的向性表型,并鉴定大脑中赋予巨噬细胞增强的向性和促融合性的信封。 J Virol 78(13):6915-6926。
  8. Peters,PJ,Sullivan,WM,Duenas-Decamp,MJ,Bhattacharya,J.,Ankghuambom,C.,Brown,R.,Luzuriaga,K.,Bell,J.,Simmonds,P.,Ball,J.and Clapham ,PR(2006)。 非巨噬细胞性人类免疫缺陷病毒1型R5包膜在血液,淋巴结和精液中占主导地位:implications for transmission and pathogenesis。 J Virol 80(13):6324-6332
  9. 这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明, Potter,EL,Chu,H.,Moore,P.,Salazar,MG,Iyer,S.,Jabara,C.,Kirchherr,J.,Mapanje,C.,Ngandu,N.,Seoighe,C.,Hoffman, I.,Gao,F.,Tang,Y.,Labranche,C.,Lee,B.,Saville,A.,Vermeulen,M.,Fiscus,S.,Morris,L.,Karim,SA,Haynes,BF ,Shaw,GM,Korber,BT,Hahn,BH,Cohen,MS,Montefiori,D.,Williamson,C.,Swanstrom,R.,Study,CAI and the Center for,HIVAVIC(2013)。 来自急性和慢性感染的病毒性Env蛋白与C型人类免疫缺陷病毒1型的比较表明糖基化和CCR5利用,并且提出了用于免疫原设计的新策略。 J Virol 87(13):7218-7233。
  10. Martin-Garcia,J.,Cao,W.,Varela-Rohena,A.,Plassmeyer,M.L.and Gonzalez-Scarano,F。(2006)。 中枢神经系统的HIV-1向性:脑源性包膜糖蛋白具有较低的CD4依赖性,并减少对融合抑制剂的敏感性。病毒学 346(1):169-179
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Joseph, S. B., Lee, B. and Swanstrom, R. (2014). Affinofile Assay for Identifying Macrophage-Tropic HIV-1. Bio-protocol 4(14): e1184. DOI: 10.21769/BioProtoc.1184.
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google 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。