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IFN-α Inhibition Assay in vitro
IFN-α 抑制的体外试验

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Abstract

During viral infections Interferon-α (IFN-α) is expressed by infected host cells. IFN-α binds to its receptor (IFNAR1/2), which leads to the activation of downstream signaling via JAK-STAT. This signaling cascade results in the expression of several hundred different genes, so called interferon-stimulated gene, which lead to an antiviral state of the infected and the neighboring cells.

Keywords: Type I IFNs(I型IFNs), IFN-alpha subtypes(IFN-α亚), Friend Murine leukemia virus(Friend Murine leukemia病毒), Antiviral effect(抗病毒作用)

Materials and Reagents

  1. Mus dunni fibroblast cells
  2. Friend murine leukemia virus (F-MuLV) (titer was measured as previously described (Robertson et al., 1991))
  3. 3-Amino-9-ethylcarbazole (AEC) (Sigma-Aldrich, catalog number: A6926-100TAB )
  4. Antibody 720 (mouse antibody against FV envelope protein), hybridoma supernatant (Robertson et al., 1991)
  5. Bovine serum albumin (BSA) (PAA Laboratories GmbH, catalog number: K41-001 )
  6. Ethanol 96% (Roth North America, catalog number: 5054.5 )
  7. Superior FBS (fetal bovine serum, not heat-inactivated) (Biochrom, catalog number: S0615 )
  8. Goat anti-mouse HRP (Dako, catalog number: P0477 )
  9. Hydrogen peroxide 30% (Applichem, catalog number: A1134,0250)
  10. N,N-dimethylformamide (Merck Millipore, catalog number: 1.03053.1000 )
  11. PBS (GIBCO, catalog number: 14190-136 )
  12. Penicillin/streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
  13. Polybrene/Hexadimethrine bromide (Sigma-Aldrich, catalog number: H9268 )
  14. RPMI 1640 (PAA Laboratories GmbH, catalog number: E15-840 )
  15. Sodium Acetate (Merck Millipore, catalog number: 1062811000 )
  16. Murine IFN-α (PBL, catalog number: 12100-1 )
  17. Medium (see Recipes)
  18. Washing Buffer (see Recipes)
  19. 3-Amino-9-ethylcarbazole (AEC) substrate solution (see Recipes)

Equipment

  1. Incubator (37 °C; 5% CO2)
  2. 24 well cell culture plate (Greiner Bio-one, catalog number: 662160 )

Procedure

Day 1:

  1. Seed 7.5 x 103 Mus dunni fibroblast cells in 500 μl media per well in 24-well plates.
  2. Add IFN-α in increasing concentrations to the cells (use concentrations between 50 pg/ml to 10,000 pg/ml).
  3. Controls: Without IFN-α; without virus.
  4. Incubate the cells for 24 h at 37 °C (5% CO2).

Day 2:

  1. Decant media.
  2. Add 1 ml fresh media supplemented with polybrene (8 μg/ml) to increase the infection efficiency.
  3. Add 50 FFU (focus-forming units) F-MuLV to the wells.
  4. Incubate for 3 days.

Day 5:

  1. Decant media.
  2. Fix cells with 500 μl 96% ethanol for 5 min at room temperature (RT).
  3. Wash wells twice with 500 μl washing buffer (PBS + 0.1% BSA).
  4. Add 250 μl supernatant of antibody 720 (mAB against FVenv) per well for 2 h.
  5. Wash twice with 500 μl washing buffer.
  6. Add 250 μl 2nd antibody (goat anti-mouse HRP) to wells (diluted 1 to 500 in PBS).
  7. Incubate for 1 h at RT.
  8. Wash twice with 500 μl washing buffer.
  9. Freshly prepare AEC substrate solution as indicated in Recipes section.
  10. Add 250 μl substrate solution per well.
  11. Incubate for 10-15 min at RT in the dark.
  12. Decant supernatant in special waste container for toxic solvents.
  13. Wash with 500 μl water.
  14. Dry plates overnight.

Day 6:

  1. Count foci
    Treatment with IFN-α should significantly decrease the numbers of foci compared to unstimulated cells (Figure 1).

Figure 1. Representative example of Interferon-α Inhibition assay with Interferon-α (left picture without foci) and without Interferon-α (right picture with foci)

Recipes

  1. Medium
    RPMI 1640
    10% FCS
    1% penicillin/streptomycin
  2. Washing buffer
    PBS + 0.1% BSA
  3. AEC substrate solution
    Dissolve 1 tablet AEC in 2.5 ml of N, N-dimethylformamide. Add 2.5 ml of the substrate solution to 47.5 ml of 50 mM sodium acetate buffer, pH 5.0. Add 25 μl of fresh 30% hydrogen peroxide immediately prior to use.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (GRK 1045).

References

  1. Dittmer, U., Brooks, D. M. and Hasenkrug, K. J. (1998). Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms. J Virol 72(8): 6554-6558. 
  2. Gibbert, K., Joedicke, J. J., Meryk, A., Trilling, M., Francois, S., Duppach, J., Kraft, A., Lang, K. S. and Dittmer, U. (2012). Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection. PLoS Pathog 8(8): e1002868.
  3. Gerlach, N., Gibbert, K., Alter, C., Nair, S., Zelinskyy, G., James, C. M. and Dittmer, U. (2009). Anti-retroviral effects of type I IFN subtypes in vivo. Eur J Immunol 39(1): 136-146.
  4. Robertson, M. N., Miyazawa, M., Mori, S., Caughey, B., Evans, L. H., Hayes, S. F. and Chesebro, B. (1991). Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting. J Virol Methods 34(3): 255-271.

简介

在病毒感染期间,干扰素-α(IFN-α)由感染的宿主细胞表达。 IFN-α结合其受体(IFNAR1/2),其导致通过JAK-STAT的下游信号传导的激活。 这种信号级联导致数百种不同基因的表达,所谓的干扰素刺激基因,其导致感染细胞和相邻细胞的抗病毒状态。

关键字:I型IFNs, IFN-α亚, Friend Murine leukemia病毒, 抗病毒作用

材料和试剂

  1. Mus dunni成纤维细胞
  2. 朋友鼠白血病病毒(F-MuLV)(如前所述测量滴度(Robertson等人,1991))
  3. 3-氨基-9-乙基咔唑(AEC)(Sigma-Aldrich,目录号:A6926-100TAB)
  4. 抗体720(针对FV包膜蛋白的小鼠抗体),杂交瘤上清液(Robertson等人,1991)
  5. 牛血清白蛋白(BSA)(PAA Laboratories GmbH,目录号:K41-001)
  6. 乙醇96%(Roth North America,目录号:5054.5)
  7. 高级FBS(胎牛血清,非热灭活)(Biochrom,目录号:S0615)
  8. 山羊抗小鼠HRP(Dako,目录号:P0477)
  9. 过氧化氢30%(Applichem,目录号:A1134,0250)
  10. N,N-二甲基甲酰胺(Merck Millipore,目录号:1.03053.1000)
  11. PBS(GIBCO,目录号:14190-136)
  12. 青霉素/链霉素(PAA Laboratories GmbH,目录号:P11-010)
  13. 聚凝胺/溴化己二甲铵(Sigma-Aldrich,目录号:H9268)
  14. RPMI 1640(PAA Laboratories GmbH,目录号:E15-840)
  15. 醋酸钠(Merck Millipore,目录号:1062811000)
  16. 小鼠IFN-α(PBL,目录号:12100-1)
  17. 中等(见配方)
  18. 洗涤缓冲液(见配方)
  19. 3-氨基-9-乙基咔唑(AEC)底物溶液(参见配方)

设备

  1. 培养箱(37℃; 5%CO 2)
  2. 24孔细胞培养板(Greiner Bio-one,目录号:662160)

程序

第1天:

  1. 种子7.5×10 3个 Mus dunni成纤维细胞在每孔500μl培养基中的24孔板中。
  2. 向细胞中加入浓度增加的IFN-α(使用浓度在50pg/ml至10,000pg/ml之间)。
  3. 对照:无IFN-α; 无病毒。
  4. 在37℃(5%CO 2)孵育细胞24小时

第2天:

  1. 清理介质。
  2. 加入1ml添加了聚凝胺(8μg/ml)的新鲜培养基以提高感染效率
  3. 向孔中添加50 FFU(聚焦形成单元)F-MuLV
  4. 孵育3天。

第5天:

  1. 清理介质。
  2. 在室温(RT)下用500μl96%乙醇固定细胞5分钟
  3. 用500μl洗涤缓冲液(PBS + 0.1%BSA)洗涤孔两次
  4. 每孔加入250μl抗体720(mAB对FVenv)的上清液2小时
  5. 用500μl洗涤缓冲液洗涤两次。
  6. 向孔中加入250μl2μL抗体(山羊抗小鼠HRP)(在PBS中稀释至1至500μl)。
  7. 在RT孵育1小时。
  8. 用500μl洗涤缓冲液洗涤两次。
  9. 如配方部分所述,新鲜制备AEC底物溶液。
  10. 每孔加入250μl底物溶液
  11. 在室温下避光孵育10-15分钟。
  12. 在特殊废物容器中倾倒上清液以备有毒溶剂
  13. 用500μl水洗涤。
  14. 干板过夜。

第6天:

  1. 计数焦点
    与未刺激的细胞相比,用IFN-α处理应显着降低病灶数目(图1)。

图1.具有干扰素-α(左图无灶)和无干扰素-α (具有灶的右图)的干扰素-α抑制试验的代表性实例

食谱

  1. 中等
    RPMI 1640
    10%FCS
    1%青霉素/链霉素
  2. 洗涤缓冲液
    PBS + 0.1%BSA
  3. AEC底物溶液
    将1片AEC溶解在2.5ml N,N-二甲基甲酰胺中。 将2.5ml底物溶液加入到47.5ml 50mM乙酸钠缓冲液(pH5.0)中。 在使用前立即加入25μl新鲜的30%过氧化氢。

致谢

这项工作得到了德意志交易所(GRK 1045)的支持。

参考文献

  1. Dittmer,U.,Brooks,D.M。和Hasenkrug,K.J。(1998)。 活减毒逆转录病毒疫苗的表征证明通过免疫机制的保护。 J Virol 72(8):6554-6558。 
  2. Gibbert,K.,Joedicke,J.J.,Meryk,A.,Trilling,M.,Francois,S.,Duppach,J.,Kraft,A.,Lang,K.S.and Dittmer, 干扰素α亚型11激活NK细胞,并能够控制逆转录病毒感染。 PLoS Pathog 8(8):e1002868。
  3. Gerlach,N.,Gibbert,K.,Alter,C.,Nair,S.,Zelinskyy,G.,James,C.M.and Dittmer,U.(2009)。 I型IFN亚型在体内的抗逆转录病毒效应 a> Eur J Immunol 39(1):136-146。
  4. Robertson,MN,Miyazawa,M.,Mori,S.,Caughey,B.,Evans,LH,Hayes,SF和Chesebro,B。(1991).a ncbi.nlm。 与变性形式的Friend鼠白血病病毒gp70包膜蛋白反应的单克隆抗体的产生:用于病灶感染性测定,免疫组织化学研究,电子显微镜和western印迹中。 /a> J Virol Methods 34(3):255-271
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gibbert, K. (2013). IFN-α Inhibition Assay in vitro. Bio-protocol 3(12): e802. DOI: 10.21769/BioProtoc.802.
  2. Gibbert, K., Joedicke, J. J., Meryk, A., Trilling, M., Francois, S., Duppach, J., Kraft, A., Lang, K. S. and Dittmer, U. (2012). Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection. PLoS Pathog 8(8): e1002868.
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