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IFN-α/β Detection Assay Using Sensor Cell Lines
感觉细胞系的α/β 干扰素检测分析   

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Abstract

Type I interferons (IFN-α/β) play an important role in host resistance to viral infections. Signaling through the JAK-STAT pathway, IFN-α/β stimulates response elements (ISRE) in the promoters of ISG to regulate their expression (reviewed in Reference 2). This method was adapted from InvivoGen to specifically detect and quantify IFN-α/β secreted in response to virus infection. HEK-Blue™ IFN-α/β cells were generated by stably introducing the human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. The activation of this pathway is made detectable by the addition of a reporter gene expressing a secreted embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG activated through an ISRE-dependent mechanism by type I IFNs.

Keywords: Interferon detection(干扰素的检测), Virus(病毒), Infection(感染), Sensor cell line(传感器细胞系), Interferon response(干扰素反应)

Materials and Reagents

  1. HEK-BlueTM IFN-α/β cells (InvivoGen, catalog number: hkb-ifnab )
  2. QUANTI-Blue (InvivoGen, catalog number: rep-qb2 )
  3. IFN-α2 (PBL Biomedical Laboratories, catalog number: PBL 11105-1 )
  4. Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA Laboratories GmbH)
  5. Fetal Calf Serum (FCS) (Biochrom)
  6. PBS (Naxo OÜ)
  7. Trypsin/EDTA (GE Healthcare)
  8. 100x Penicillin-Streptomycin (Naxo OÜ)
  9. Normocin (InvivoGen, catalog number: ant-nr-1 )
  10. Zeocin (InvivoGen, catalog number: ant-zn-1 )
  11. Blasticidin (Sigma-Aldrich, catalog number: 3513-03-9 )

Equipment

  1. 96-well plate
  2. 37 °C, 5% CO2 cell culture incubator
  3. Microscope
  4. UV cross-linker (Hoefer, model: UVC5000 )
  5. Tecan SunriseTM microplate reader

Procedure

  1. Cells and media
    HEK-BlueTM IFN-α/β cells were maintained in DMEM containing 10% heat-inactivated FCS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 100 μg/ml Normocin in a humidified incubator at 37 °C under 5% CO2. HEK-BlueTM IFN-α/β cells should not be passaged more than 20 times to remain fully efficient. HEK-BlueTM IFN-α/β cells should be maintained with two selective antibiotics, Zeocin (100 mg/ml) and Blasticidin (30 μg/ml). Do not use selective antibiotics for the test procedure. Cells should be passaged when a 70-80% confluency is reached.

  2. Interferon detection
    Day 1
    1. Add 50 µl of each sample per well of a flat-bottom 96-well plate. If infectious virus is present in the collected samples, it should be inactivated using UV cross-linker for 5 min at 1,000 μJ/cm2.
      The sample of the 96-well plate with standards, samples and negative controls (see next steps for the details). “Mock” indicates the same amount of media, but from uninfected cells. “Neg.c.” indicates negative control and described below.

      IFN (1)       
      IFN (1)     
      IFN (1)   
      Sample 1   
      Sample 1   
      Sample 1
                
                 
                
                
                 
                 
      IFN (2)   
      IFN (2)   
      IFN (2)   
      Sample 2  
      Sample 2   
      Sample 2






      IFN (3)    
      IFN (3)  
      IFN (3)   
      Sample 3   
      Sample 3   
      Sample 3






      IFN (4)     
      IFN (4)   
      IFN (4)   
      Sample 4   
      Sample 4  
      Sample 4






      IFN (5)    
      IFN (5)   
      IFN (5)   
      Sample 5   
      Sample 5   
      Sample 5






      IFN (6)   
      IFN (6)   
      IFN (6)   
      Mock  
      Mock   
      Mock






      IFN (7)  
      IFN (7)   
      IFN (7)   
      Neg.c. 1  
      Neg.c. 1   
      Neg.c. 1






      IFN (8)   
      IFN (8)   
      IFN (8)   
      Neg.c. 2  
      Neg.c. 2   
      Neg.c. 2






           
    2. Add 50 µl of IFN-α or IFN-β dilutions (1-8).

      1   
      2   
      3   
      4   
      5   
      6   
      7   
      8
      10,000 U/ml   
      3,333 U/ml   
      1,111 U/ml   
      370 U/ml   
      123 U/ml   
      41 U/ml   
      13.7 U/ml   
      4.6 U/ml
      75 µl of 104 IU/ml   
      50 µl DMEM   
      50 µl DMEM   
      50 µl DMEM   
      50 µl DMEM   
      50 µl DMEM   
      50 µl DMEM   
      50 µl DMEM

      Transfer 25 µl to each next well to make 1:3 dilutions as demonstrated in the table above. To prepare the dilutions, use the same DMEM as for the maintenance of HEK-BlueTM IFN-α/β cells (no selective antibiotics needed for this step).
    3. Suggested negative controls: (1) 50 µl of IFN-γ at 104 U/ml in one well, (2) 50 µl of UV-inactivated medium from virus infected IFN-α/β-negative cell line (like BHK-21) with titer of virus similar to samples used in the same experiment.
    4. Prepare suspension of HEK-BlueTM IFN-α/β cells at ~280,000 cells per ml in test medium (DMEM containing 10% heat-inactivated FCS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 100 μg/ml Normocin).
    5. Add 180 µl of cell suspension (~50,000 cells) per well.
    6. Incubate the plate at 37 °C in a CO2 incubator for 20-24 h.

    Day 2
    1. Prepare QUANTI-BlueTM reagent following the instructions on the package.
    2. Add 180 µl of resuspended QUANTI-BlueTM reagent per well of a flat-bottom 96-well plate.
    3. Add 50 µl of supernatant from induced HEK-BlueTM IFN-α/β cells.
    4. Incubate the plate for 1-3 h at 37 °C in CO2 incubator.
    5. Determine SEAP levels using a spectrophotometer (Tecan Sunrise plate reader) at 620-655 nm.
    6. Calculations should be performed using plots drawn separately for each range of obtained values. Please note that the curve is not linear and IFN values should be within the detection range, otherwise use dilutions of initial samples.
      Representative curve obtained in one of the experiments is shown below. Please note that in given experimental conditions higher concentrations of IFN (1,000-10,000) reach the plateau level and dilutions of initial samples are strongly recommended:



      Note: manufacturer recommends using 20 µl of sample volume (day 1) and 20 µl of supernatant (day 2), but we found that increased volumes (50 µl) improved the reproducibility of the results.

Notes

  1. All specific product information and guidelines can be found at the manufacturer’s website:
    http://www.invivogen.com/hek-blue-ifn-ab.
  2. Cell line stability
    It is critical to prepare an adequate number of frozen stocks at early passages. HEK-BlueTM IFN-α/β cells should not be passaged more than 20 times to remain fully efficient. HEK-BlueTM IFN-α/β cells should be maintained in growth medium supplemented with the two selective antibiotics, ZeocinTM (100 mg/ml) and Blasticidin (30 μg/ml). Antibiotic pressure with Blasticidin is required to maintain the plasmid coding for human STAT2 and IRF9 genes, and ZeocinTM is required to maintain the plasmid coding for SEAP.

Acknowledgments

This protocol was published in the original paper by Lulla et al. (2013). This work was supported by Estonian Science Foundation grants 7501, 7407, and9421; target financing project SF0180087s08; and the European Union through the European Regional Development Fund via the Center of Excellence in Chemical Biology.

References

  1. Lulla, V., Karo-Astover, L., Rausalu, K., Merits, A. and Lulla, A. (2013). Presentation overrides specificity: probing the plasticity of alphaviral proteolytic activity through mutational analysis. J Virol 87(18): 10207-10220.
  2. Randall, R. E. and Goodbourn, S. (2008). Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89(Pt 1): 1-47.

简介

I型干扰素(IFN-α/β)在宿主对病毒感染的抗性中起重要作用。 通过JAK-STAT途径的信号传导,IFN-α/β刺激ISG启动子中的反应元件(ISRE)以调节它们的表达(参见参考文献2)。 此方法改编自InvivoGen以特异性检测和量化响应于病毒感染分泌的IFN-α/β。 通过将人STAT2和IRF9基因稳定地引入HEK293细胞中以产生完全活性的I型IFN信号传导途径,产生HEK-Blue TM IFN-α/β细胞。 通过加入在ISG54启动子控制下表达分泌性胚胎碱性磷酸酶(SEAP)的报告基因,可以检测该途径的活化。 ISG54是通过I型IFN的ISRE依赖性机制激活的公知的ISG

关键字:干扰素的检测, 病毒, 感染, 传感器细胞系, 干扰素反应

材料和试剂

  1. HEK-Blue TM/IFN-α/β细胞(InvivoGen,目录号:hkb-ifnab)
  2. QUANTI-Blue(InvivoGen,目录号:rep-qb2)
  3. IFN-α2(PBL Biomedical Laboratories,目录号:PBL 11105-1)
  4. Dulbecco's Modified Eagle's Medium(DMEM)(PAA Laboratories GmbH)
  5. 胎牛血清(FCS)(Biochrom)
  6. PBS(NaxoOÜ)
  7. 胰蛋白酶/EDTA(GE Healthcare)
  8. 100x青霉素 - 链霉素(NaxoOÜ)
  9. Normocin(InvivoGen,目录号:ant-nr-1)
  10. Zeocin(InvivoGen,目录号:ant-zn-1)
  11. 杀稻瘟素(Sigma-Aldrich,目录号:3513-03-9)

设备

  1. 96孔板
  2. 37℃,5%CO 2细胞培养箱中培养
  3. 显微镜
  4. UV交联剂(Hoefer,型号:UVC5000)
  5. Tecan Sunrise TM 酶标仪

程序

  1. 单元格和媒体
    将HEK-Blue TM sup/TM IFN-α/β细胞维持在含有10%热灭活的FCS,50U/ml青霉素,50μg/ml链霉素和100μg/ml Normocin的DMEM中的加湿 在37℃,5%CO 2培养箱中。 HEK-Blue TM IFN-α/β细胞不应传代超过20次以保持完全有效。 应该用两种选择性抗生素Zeocin(100mg/ml)和杀稻瘟素(30μg/ml)维持HEK-Blue TM sup/TM IFN /α/β细胞。 在测试过程中不要使用选择性抗生素。 当达到70-80%汇合时,应传代细胞。

  2. 干扰素检测
    第1天
    1. 每孔加入50μl平底96孔板的每个样品。 如果感染性病毒存在于所收集的样品中,则应该使用UV交联剂在1,000μJ/cm 2下将其灭活5分钟。 96孔板的样品用标准品,样品和阴性对照(详见后续步骤)。 "模拟"表示相同量的培养基,但来自未感染的细胞。 "Negc.c."表示阴性对照,如下所述
      IFN(1)       
      IFN(1)     
      IFN(1)   
      示例1   
      示例1   
      示例1
                
                 
                
                
                 
                 
      IFN(2)   
      IFN(2)   
      IFN(2)   
      示例2  
      示例2   
      示例2






      IFN(3)    
      IFN(3)  
      IFN(3)   
      示例3   
      示例3   
      示例3






      IFN(4)     
      IFN(4)   
      IFN(4)   
      示例4   
      示例4  
      示例4






      IFN(5)    
      IFN(5)   
      干扰素(5)   
      示例5   
      示例5   
      示例5






      IFN(6)   
      IFN(6)   
      IFN(6)   
      Mock  
      Mock   
      模拟






      IFN(7)  
      IFN(7)   
      IFN(7)   
      负电压 1  
      负电压 1   
      负电压 1






      IFN(8)   
      IFN(8)   
      IFN(8)   
      负电压 2  
      负电压 2   
      负电压 2






           
    2. 加入50μlIFN-α或IFN-β稀释液(1-8)。

      1   
      2   
      3   
      4   
      5   
      6   
      7   
      8
      10,000 U/ml   
      3,333 U/ml   
      1,111 U/ml   
      370 U/ml   
      123 U/ml   
      41 U/ml   
      13.7 U/ml   
      4.6 U/ml
      75μl的10 4 IU/ml  
      50μlDMEM   
      50μlDMEM   
      50μlDMEM   
      50μlDMEM   
      50μlDMEM   
      50μlDMEM   
      50微升DMEM

      转移25微升到每个下一个井,使1:3稀释,如上表所示。 为了制备稀释液,使用与用于维持HEK-Blue TM IFN-α/β细胞(此步骤不需要选择性抗生素)相同的DMEM。
    3. 建议的阴性对照:(1)50μl在10 4 U/ml的IFN-γ在一个孔中,(2)50μl来自病毒感染的IFN-α/β-阴性的UV-灭活培养基 细胞系(如BHK-21),其具有与在相同实验中使用的样品相似的病毒滴度
    4. 在测试培养基(含有10%热灭活的FCS,50U/ml青霉素,50μg/ml链霉素的DMEM)中制备HEK-Blue TM IFN-α/β细胞以约280,000个细胞/,和100μg/ml诺莫菌素)
    5. 每孔加入180μl细胞悬浮液(〜50,000个细胞)
    6. 将板在CO 2培养箱中在37℃下孵育20-24小时。

    第2天
    1. 按照包装上的说明准备QUANTI-Blue TM 试剂
    2. 向平底96孔板的每孔中加入180μl重悬的QUANTI-Blue TM sup试剂。
    3. 加入50μl来自诱导的HEK-Blue TM IFN-α/β细胞的上清液
    4. 在37℃下在CO 2培养箱中孵育平板1-3小时
    5. 使用分光光度计(Tecan Sunrise读板器)在620-655nm测定SEAP水平
    6. 应使用对于所获得的值的每个范围单独绘制的绘图进行计算。 请注意,曲线不是线性的,IFN值应在检测范围内,否则使用 初始样品的稀释液 在一个实验中获得的代表性曲线如下所示。 请注意,在给定的实验条件下,高浓度的IFN(1,000-10,000)达到平台水平,强烈建议稀释初始样品:



      注意:制造商建议使用20μl样品体积(第1天)和20μl上清液(第2天),但是我们发现增加体积(50μl)提高了结果的重现性。

笔记

  1. 所有具体的产品信息和指南可以在制造商的网站上找到:
    http://www.invivogen.com/hek-blue-ifn-ab.
  2. 细胞系稳定性
    在早期阶段准备足够数量的冷冻储备物是至关重要的。 HEK-Blue TM IFN-α/β细胞不应传代超过20次以保持完全有效。 HEK-Blue TM/IFN-α/β细胞应保持在补充有两种选择性抗生素Zeocin TM(100mg/ml)和杀稻瘟素(30μg)的生长培养基中/ml)。需要使用杀稻瘟素的抗生素压力来维持编码人STAT2和IRF9基因的质粒,并且需要Zeocin TM 来维持编码SEAP的质粒。

致谢

该协议发表在Lulla等人的原始论文中。(2013)。这项工作得到爱沙尼亚科学基金会拨款7501,7407和9421的支持;目标融资项目SF0180087s08;和欧洲联盟通过欧洲区域发展基金通过化学生物学卓越中心。

参考文献

  1. Lulla,V.,Karo-Astover,L.,Rausalu,K.,Merits,A.and Lulla,A.(2013)。 演示文稿覆盖了特异性:通过突变分析探测甲病毒蛋白水解活性的可塑性。 J Virol 87(18):10207-10220。
  2. Randall,R.E.and Goodbourn,S。(2008)。 干扰素和病毒:诱导,信号传导,抗病毒反应和病毒对策之间的相互作用。 J Gen Virol 89(Pt 1):1-47。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lulla, V., Merits, A. and Lulla, A. (2014). IFN-α/β Detection Assay Using Sensor Cell Lines. Bio-protocol 4(10): e1130. DOI: 10.21769/BioProtoc.1130.
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