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Measurement of IFN-α Subtype Concentrations (Virus-free, Cell-based Bioassay)
IFN-α亚型浓度的测定(无病毒,基于细胞的生物分析)   

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Abstract

The induction of type I IFN is the immediate host response against viral infections. Type I IFNs belong to a multigene family including up to 14 different IFN-α subtypes and one IFN-β. They are highly conserved and bind the same receptor (IFNAR1/2) with varying affinities, although they differ in their biological activities.

Keywords: Type I IFNs(I型IFNs), IFN-alpha subtypes(IFN-α亚), Mx expression(MX的表达)

Materials and Reagents

  1. 7AAD (7-amino-actinomycin D) (BD Pharmingen, catalog number: 51-68981E )
  2. Bovine serum albumin (BSA) (PAA Laboratories GmbH, catalog number: K41-001 )
  3. DMEM (Life Technologies, Gibco®, catalog number: 41966-029 )
  4. Superior FBS (fetal bovine serum, not heat-inactivated) (Biochrom, catalog number: S0615 )
  5. Mx/RAGE7 cells (virus-transformed adherent cell line with a temperature-inducible promotor; must be cultured at 32 °C; cells express the Mx transgene and a promotorless eGFP gene which is expressed due to type I IFN stimulation ) (Bollati-Fogolin and Muller, 2005)
  6. PBS (Life Technologies, Gibco®, catalog number: 14190-136 )
  7. Penicillin/streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
  8. Propidium iodide (eBioscience, catalog number: 00-6990-50 )
  9. Murine IFN-α (PBL, catalog number: 12100-1 )
  10. Sodium azide (Applichem, catalog number: A1430.0010 )
  11. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360-039 )
  12. Trypsin EDTA (PAA Laboratories GmbH, catalog number: L11-004 )
  13. β-mercaptoethanol (Life Technologies, Gibco®, catalog number: 31350-010 )
  14. Media for Mx/RAGE7 cells (see Recipes)
  15. FACS buffer (see Recipes)

Equipment

  1. 96-well flat bottom plate (Falcon BD Labware, catalog number: 3072 )
  2. 1.5 ml microfuge tubes
  3. FACS tubes (BD Biosciences, Falcon®, catalog number: 352054 )
  4. Flow cytometer (e.g. BD LSR II)
  5. Incubator (37 °C; 5% CO2)
  6. Incubator (32 °C; 5% CO2)

Procedure

Different murine IFN-α subtypes (IFN-α1, -α2, -α4, -α5, -α6, -α9, -α11) were produced as already described (Gerlach et al., 2009).


Day 1:

  1. Seed Mx/RAGE7 cells in a 96 well cell culture plate (2 x 104 cells per well in 200 μl medium).
  2. Grow the cells for 24 h at 32 °C.

Day 2:

  1. Perform serial dilutions (log10) of produced IFN-α subtypes in medium in 1.5 ml tubes.
  2. Perform serial dilutions (log2) of recombinant IFN-α subtypes (PBL) with known concentrations from 1,000 U/ml to 31.25 U/ml (= standards) in 1.5 ml tubes.
  3. Decant medium of Mx/RAGE7 cells.
  4. Add 200 μl of the IFN-α solutions with known (standards) and unknown concentrations to the cells.
  5. As negative control add 200 μl of medium without IFN-α.
  6. Incubate the samples for 24 h at 37 °C.

Day 3:

  1. Decant the medium.
  2. Add 200 μl fresh medium to the cells.
  3. Incubate the samples for 48 h at 37 °C.

Day 5:

  1. Decant the medium.
  2. Wash cells with 200 μl PBS.
  3. Add 50 μl of trypsin EDTA (1x) 0.05% to the cells at room temperature until they suspend.
  4. Harvest suspended cells in FACS tubes containing 1 ml of PBS.
  5. Centrifuge cells (300 x g; 5 min).
  6. Resuspend cells with 250 μl FACS buffer.
  7. Add 2.5 μl 7AAD or 0.5 μl propidium iodide per sample to exclude dead cells.
  8. Immediately analyze cells with flow cytometer.
  9. IFN-α treated Mx/RAGE7 cells express eGFP (Figure 1).
  10. Perform standard curve with samples treated with known IFN-α concentrations (graph the data for the standard curve (Figure 2), the IFN-α titer can be determined by comparison).
  11. Calculate concentrations of unknown samples.


Figure 1. Representative dot plots of Mx/RAGE7 cells without IFN-α (upper panel) and with IFN-α (lower panel)

Figure 2. Standard curve of IFN-α

Recipes

  1. Media for Mx/RAGE7 cells
    DMEM
    10% FBS
    1 mM sodium pyruvate
    1% penicillin/streptomycin
    50 μM β-mercaptoethanol
  2. FACS buffer
    PBS
    0.1% BSA
    0.02% sodium azide

Acknowledgments

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

References

  1. Bollati-Fogolin, M. and Muller, W. (2005). Virus free, cell-based assay for the quantification of murine type I interferons. J Immunol Methods 306(1-2): 169-175.
  2. 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.
  3. 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.

简介

I型IFN的诱导是针对病毒感染的直接宿主应答。 I型IFNs属于多基因家族,包括多达14种不同的IFN-α亚型和一种IFN-β。 它们是高度保守的,并以不同的亲和力结合相同的受体(IFNAR1/2),尽管它们的生物活性不同。

关键字:I型IFNs, IFN-α亚, MX的表达

材料和试剂

  1. 7AAD(7-氨基 - 放线菌素D)(BD Pharmingen,目录号:51-68981E)
  2. 牛血清白蛋白(BSA)(PAA Laboratories GmbH,目录号:K41-001)
  3. DMEM(Life Technologies,Gibco ,目录号:41966-029)
  4. 高级FBS(胎牛血清,非热灭活)(Biochrom,目录号:S0615)
  5. Mx/RAGE7细胞(具有温度诱导型启动子的病毒转化的贴壁细胞系;必须在32℃下培养;细胞表达Mx转基因和由于I型IFN刺激而表达的无启动子的eGFP基因)(Bollati-Fogolin 和Muller,2005)
  6. PBS(Life Technologies,Gibco ,目录号:14190-136)
  7. 青霉素/链霉素(PAA Laboratories GmbH,目录号:P11-010)
  8. 碘化丙啶(eBioscience,目录号:00-6990-50)
  9. 小鼠IFN-α(PBL,目录号:12100-1)
  10. 叠氮化钠(Applichem,目录号:A1430.0010)
  11. 丙酮酸钠(Life Technologies,Gibco ,目录号:11360-039)
  12. 胰蛋白酶EDTA(PAA Laboratories GmbH,目录号:L11-004)
  13. β-巯基乙醇(Life Technologies,Gibco ,目录号:31350-010)
  14. Mx/RAGE7单元的介质(参见配方)
  15. FACS缓冲区(参见配方)

设备

  1. 96孔平底板(Falcon BD Labware,目录号:3072)
  2. 1.5 ml微量离心管
  3. FACS管(BD Biosciences,Falcon ,目录号:352054)
  4. 流式细胞仪(例如 BD LSR II)
  5. 培养箱(37℃; 5%CO 2)
  6. 培养箱(32℃; 5%CO 2)

程序

如已经描述的(Gerlach等人,2009)产生了不同的鼠IFN-α亚型(IFN-α1,-α2,-α4,-α5,-α6,-α9,-α11)。


第1天:

  1. 种子Mx/RAGE7细胞在96孔细胞培养板中(在200μl培养基中每孔2×10 4个细胞)。
  2. 在32℃下培养细胞24小时。

第2天:

  1. 在1.5ml试管中在培养基中进行生产的IFN-α亚型的系列稀释(log10)
  2. 在1.5ml试管中,用1,000U/ml至31.25U/ml(=标准品)的已知浓度进行连续稀释(log2)重组IFN-α亚型(PBL)。
  3. 稀释培养基Mx/RAGE7细胞
  4. 添加200微升已知(标准)和未知浓度的IFN-α溶液到细胞中
  5. 作为阴性对照,加入200μl无IFN-α的培养基
  6. 在37℃下孵育样品24小时

第3天:

  1. 滗析培养基。
  2. 向细胞中加入200μl新鲜培养基
  3. 在37℃下孵育样品48小时

第5天:

  1. 滗出介质。
  2. 用200μlPBS洗涤细胞
  3. 在室温下向细胞中加入50μl胰蛋白酶EDTA(1x)0.05%,直到它们悬浮
  4. 收获悬浮细胞在含有1ml PBS的FACS管
  5. 离心细胞(300×g; 5分钟)
  6. 用250μlFACS缓冲液重悬细胞
  7. 每个样品加入2.5μl7AAD或0.5μl碘化丙啶以排除死细胞
  8. 立即用流式细胞仪分析细胞
  9. IFN-α处理的Mx/RAGE7细胞表达eGFP(图1)。
  10. 对用已知IFN-α浓度处理的样品进行标准曲线(图2),可以通过比较来确定标准曲线的数据(图2),IFN-α滴度。
  11. 计算未知样品的浓度。


图1.没有IFN-α(上图)和IFN-α(下图)的Mx/RAGE7细胞的代表性点图

图2. IFN-α的标准曲线

食谱

  1. Mx/RAGE7单元的媒体
    DMEM
    10%FBS
    1mM丙酮酸钠 1%青霉素/链霉素 50μMβ-巯基乙醇
  2. FACS缓冲区
    PBS
    0.1%BSA
    0.02%叠氮化钠

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. Bollati-Fogolin,M。和Muller,W。(2005)。 Virus without,cell-based assay for the quantification of murine type I interferons. Immunol Methods 306(1-2):169-175。 br />
  2. 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。
  3. 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。
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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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gibbert, K. (2013). Measurement of IFN-α Subtype Concentrations (Virus-free, Cell-based Bioassay). Bio-protocol 3(12): e803. DOI: 10.21769/BioProtoc.803.
  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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