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Vaccine-induced Cytokine Production Detected by Luminex Multiplex Analysis
Lunimex 多因子分析试剂盒检测疫苗诱导的细胞因子的生成   

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参见作者原研究论文

本实验方案简略版
Cancer Research
Nov 2013

Abstract

Different vaccine and adjuvant combinations are known to rapidly induce antigen presenting cell (APC) maturation and pro-inflammatory cytokine and production, which in turn play an important role in the priming of antigen-specific T cells. Measuring cytokine production systemically in the serum fails to detect localized responses in the lymph nodes draining a subcutaneous immunization site. On the other hand, stimulating APC with vaccine formulations in vitro lacks the complexity of the lymph node microenvironment and the presence of other in vivo factors. Here we analyse cytokine production directly in vaccine draining lymph nodes (dLN) extracted early after in vivo vaccination. To do this we perform cytokine multiplex analysis of supernatants from whole dLN cell suspensions following a brief ex vivo incubation.

Materials and Reagents

  1. C57BL/6 mice from Harlan Laboratories (the Netherlands)
    Note: All mice used in this protocol were between 6 and 12 weeks of age and were sex and age matched for each individual experiment. Three mice were used per group in each experiment.
  2. OVA257-264 and OVA323-339 peptides
    Note: Peptides were manufactured by the Protein and Peptide Chemistry Facility (PPCF) of the University of Lausanne.
  3. Iscove's modified Dulbecco's medium (IMDM), GlutaMAX™ supplement (Life Technologies, Gibco®, catalog number: 31980-030 )
  4. Penicillin-Streptomycin (5,000 U/ml) (Life Technologies, Gibco®, catalog number: 15070063 )
  5. 2-Mercaptoethanol (2-ME, 55 mM) (Life Technologies, Gibco®, catalog number: 21985-023 )
  6. Fœtal bovine serum (FBS) (performance sera with low endotoxin: qualified, US origin) (Life Technologies, catalog number: 26140 or similar)
  7. Phosphate Buffered Saline (Laboratorium Dr. Bichsel AG)
  8. Poly (I: C) HMW (tlrl-pic) and Imiquimod R837 (tlrl-imq, InvivoGen)
  9. CpG-ODN 1826 (Coley Pharmaceuticals. No longer available. CpG-ODN 1826, tlrl-1826 from InvivoGen can be substitued)
  10. Quil A saponin mix from Quillaja saponaria (Brenntag Nordic A/S)
  11. Cytokine Mouse 10-Plex Panel for Luminex platform (Life Technologies, Novex®, catalog number: LMC0001 ) for use with the Luminex® 100™⁄200™ and FLEXMAP 3D® systems
    User manual available online: http://tools.lifetechnologies.com/content/sfs/manuals/LMC0001_Protocol_Rev1.pdf
  12. Complete iscove's modified Dulbecco's medium (cIMDM) (see Recipes)

Equipment

  1. 1 ml BD Tuberculin Syringe & 26 g or 27 g x 0.5" BD™ PrecisionGlide needle (Beckton Dickinson, catalog numbers: 3052111 or 305109 )
  2. Falcon™ 24 well, non-treated, flat-bottom tissue culture plates (Corning, catalog number: 351147 )
  3. ~40 μm nylon gauze sheets cut into small squares, e.g. Nylon Mesh Lab Pak, 41 Microns Square Opening (Nitex, catalog number: 7050-1220-000-14 ) or Nylon 6 Mesh Sheet, 48 microns Mesh Size (Small Parts, catalog number: B0043D1SCE )
  4. Plunger from 1ml BD™ Tuberculin Syringe (BD, catalog number: 309602 )
  5. Dissection scissors and fine-nosed forceps or tweezers
  6. Falcon™ 96 well, non-treated, U-bottom tissue culture plates (Corning, catalog number: 351177 )
  7. Luminex® 200™ System with xPONENT® Software (Life Technologies, Novex®)

Software

  1. xPONENT® software (Life Technologies, Novex®)
  2. Statistical tests were performed using GraphPad Prism software
    Note: The different groups were compared using One-way ANOVA with the Dunnett multiple comparison test, comparing all groups to the peptide alone group.

Procedure

  1. Experimental protocol
    1. C57BL/6 mice were immunised with 10 μg OVA257-264 and 10 μg OVA323-339 peptides in 100 μl PBS subcutaneously at the base of the tail: Briefly, mice were restrained in a holding tunnel with an opening at the end to access the base of the tail. About half the length of the needle of the tuberculin syringe was inserted into the subcutaneous layer of skin just above and to the side of the tail. The correct depth can be confirmed by visualizing the bevel just below the skin. The vaccine is slowly injected while keeping pressure at the needle entry point. A bubble of liquid should become visible under the skin. The needle should be withdrawn carefully and the exit point massaged slightly with the fingertip to prevent escape of the vaccine.
    2. Peptides were injected alone or in combination with 50 μg of one of the following adjuvants: CpG-ODN (CpG), HMW Poly (I: C), imiquimod, or Quil A.
    3. Either 12 or 24 h later, mice were euthanised by CO2 asphyxiation followed by cervical dislocation, and dLN (both inguinal LN) were excised.
    4. LNs were rapidly transferred to a 24 well plate containing 250 μl of pre-warmed cIMDM + 5% FBS over a small square of 45 μm nylon gauze. A second piece of gauze was placed on top of the LN tissue, which was then dissociated by gently crushing with the rubber end of a 1 ml syringe plunger, while holding the mesh squares in place with forceps.
    5. The cell suspension was then immediately pipetted into a 96 well u-bottom plate for incubation at 37 degrees for 1, 6 or 12 h.
    6. The plate was centrifuged at 300 x g for 1 min before removing 65 μl of supernatant and freezing it at -20 degrees for later analysis.
      Note: Samples collected from multiple time-points for combined analysis should always be frozen rapidly to preserve the cytokines. However, analysis can be performed immediately if only a single time-point is to be analysed.

  2. Luminex analysis
    1. Cytokine production was measured with the mouse 10-plex Luminex panel [granulocyte macrophage colony–stimulating factor (GM-CSF), IFN-γ, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70 and TNF-α], and analysed using the Luminex 200 system with xPONENT software.
    2. On the day of analysis, samples were thawed at room temperature.
    3. The protocol outlined in the Luminex manual was followed exactly.
    4. Standards were reconstituted with a mixture composed of 50% assay diluent and 50% of cIMDM.
    5. 50 μl of thawed sample was added to the luminex plate along with 50 μl of assay diluent during the analyte capture phase.
    6. The samples were run on the Luminex® 200™ system with xPONENT® software according to the manufacturer’s instructions.

Recipes

  1. Complete medium (cIMDM)
    IMDM
    100 U/ml Penicillin & 100 μg/ml Streptomycin
    55 μM 2-ME
    5% FBS

Acknowledgments

Abbreviated protocol previously published in: Perret et al. (2013). This work was supported by grants from the New Zealand Foundation for Research Science and Technology and the Emma Muschamp Foundation (R. Perret) and from the Swiss National Science Foundation (310030-130812 and CRSII3_141879) and the Medic Foundation (P. Romero). Disclosure of Potential Conflicts of Interest: 
P. Romero is a consultant/advisory board member of Immatics Biotechnologies, DC Prime, Matwin, and Center for Human Immunology, Pasteur Institute (Paris, France). No potential conflicts of interest were disclosed by the other authors.

References

  1. Perret, R., Sierro, S. R., Botelho, N. K., Corgnac, S., Donda, A. and Romero, P. (2013). Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity. Cancer Res 73(22): 6597-6608.

简介

已知不同的疫苗和佐剂组合快速诱导抗原呈递细胞(APC)成熟和促炎细胞因子和产生,其反过来在抗原特异性T细胞的引发中起重要作用。 在血清中全身测量细胞因子产生不能检测排出皮下免疫位点的淋巴结中的局部反应。 另一方面,用疫苗制剂在体外刺激APC缺少淋巴结微环境的复杂性和其他体内因子的存在。 在这里我们分析细胞因子生产直接在疫苗引流淋巴结(dLN)提前早期在体内接种。 为此,我们在短暂的离体孵育后对来自整个dLN细胞悬浮液的上清液进行细胞因子多重分析。

材料和试剂

  1. C57BL/6小鼠(来自Harlan Laboratories(荷兰)) 注意:在该方案中使用的所有小鼠在6-12周龄之间,并且对于每个个体实验是性别和年龄匹配的。 每个实验中每组使用三只小鼠。
  2. OVA257-264和OVA323-339肽
    注意:肽由洛桑大学的蛋白质和肽化学设备(PPCF)制造。
  3. Iscove改良的Dulbecco培养基(IMDM),GlutaMAX TM补充剂(Life Technologies,Gibco ,目录号:31980-030)
  4. 青霉素 - 链霉素(5,000U/ml)(Life Technologies,Gibco ,目录号:15070063)
  5. 2-巯基乙醇(2-ME,55mM)(Life Technologies,Gibco ,目录号:21985-023)
  6. 牛血清(FBS)(具有低内毒素的性能血清:合格,来源于美国)(Life Technologies,目录号:26140或类似物)
  7. 磷酸盐缓冲盐水(Laboratorium Dr.Bichsel AG)
  8. 聚(I:C)HMW(tlrl-pic)和咪喹莫特R837(tlrl-imq,InvivoGen)
  9. CpG-ODN 1826(Coley Pharmaceuticals,不再可用,CpG-ODN 1826,来自InvivoGen的tlrl-1826可以被取代)
  10. 来自Quillaja saponaria(Brenntag Nordic A/S)的Quil A皂苷混合物
  11. 用于Luminex平台(Life Technologies,Novex ,目录号:LMC0001)的细胞因子鼠标10-Plex面板,用于Luminex 100/200 TM和FLEXMAP 3D < sup>®系统
    用户手册可在线查看: http://tools.lifetechnologies.com/content/sfs/手册/LMC0001_Protocol_Rev1.pdf
  12. 完成iscove的修改的杜尔贝科介质(cIMDM)(参见配方)

设备

  1. 1ml BD Tuberculin Syringe& 26g或27g×0.5"BD TM PrecisionGlide针(Beckton Dickinson,目录号:3052111或305109)
  2. Falcon TM 24孔,未处理的平底组织培养板(Corning,目录号:351147)
  3. 〜40μm尼龙纱布片材切割成小正方形,例如Nylon Mesh Lab Pak,41微米正方形开口(Nitex,目录号:7050-1220-000-14)或尼龙6网片,48微米 网格尺寸(小零件,目录号:B0043D1SCE)
  4. 来自1ml BD TM结核菌素注射器(BD,目录号:309602)的柱塞
  5. 解剖剪刀和细镊子或镊子
  6. Falcon TM 96孔,未处理的U形组织培养板(Corning,目录号:351177)
  7. 具有xPONENT ®软件(Life Technologies,Novex ®)的Luminex ® 200™系统

软件

  1. xPONENT ®软件(Life Technologies,Novex ®
  2. 使用GraphPad Prism软件
    进行统计学测试 注意:使用单因素方差分析与Dunnett多重比较检验比较所有组与单独肽组的不同组。

程序

  1. 实验方案
    1. 在尾巴的基部,用100μlPBS中的10μgOVA 257-264和10μgOVA 323-339肽对C57BL/6小鼠皮下免疫。简而言之,将小鼠被限制在具有端部开口的保持隧道中以接近尾部的基部。将结核菌素注射器的针的大约一半长度插入到刚好在尾部上方和侧面的皮肤的皮下层中。正确的深度可以通过可视化皮肤下方的斜角来确认。在针入口点保持压力的同时缓慢注射疫苗。液体泡应该在皮肤下可见。应小心取出针头,并用指尖轻轻按摩出口点,以防疫苗逃逸。
    2. 肽单独注射或与50μg下列佐剂之一组合注射:CpG-ODN(CpG),HMW Poly(I:C),咪喹莫特或Quil.
    3. 在12或24小时后,通过CO 2窒息,随后颈脱位将小鼠安乐死,切除dLN(均为腹股沟淋巴结)。
    4. 将LN迅速转移到含有250μl预热的cIMDM + 5%FBS的24孔板中,在小方形的45μm尼龙纱布上。将第二块纱布放置在LN组织的顶部,然后通过用1ml注射器柱塞的橡胶端轻轻压碎,同时用镊子将网格保持在适当的位置而解离。
    5. 然后将细胞悬浮液立即移液到96孔u底板中,在37℃孵育1,6或12小时。
    6. 将平板在300×g离心1分钟,然后除去65μl上清液,并在-20度冷冻以备后续分析。
      注意:从多个时间点收集的用于组合分析的样品应该总是快速冷冻以保存细胞因子。但是,如果只分析一个时间点,则可立即进行分析。

  2. Luminex分析
    1. 细胞因子产生用小鼠10-fold Luminex组[粒细胞巨噬细胞集落刺激因子(GM-CSF),IFN-γ,IL-1b,IL-2,IL-4,IL-5,IL-6,IL- -10,IL-12p40/p70和TNF-α],并使用具有xPONENT软件的Luminex 200系统进行分析。
    2. 在分析当天,将样品在室温下解冻
    3. 在Luminex手册中概述的协议是精确遵循的。
    4. 用由50%测定稀释剂和50%cIMDM组成的混合物重建标准品
    5. 在分析物捕获阶段期间,将50μl解冻样品与50μl测定稀释剂一起加入luminex平板中。
    6. 根据制造商的说明书,在具有xPONENT软件的Luminex?系统上运行样品。

食谱

  1. 完整媒体(cIMDM)
    IMDM
    100U/ml青霉素& 100μg/ml链霉素 55μM2-ME
    5%FBS

致谢

以前发布的简称协议:Perret等人。(2013)。这项工作得到了新西兰研究科学与技术基金会和Emma Muschamp基金会(R. Perret)和瑞士国家科学基金会(310030-130812和CRSII3_141879)和Medic基金会(P. Romero)的资助。披露潜在的利益冲突:P. Romero是Immatics Biotechnologies,DC Prime,Matwin和巴斯德研究所(巴黎,法国)人类免疫中心的顾问/顾问委员会成员。其他作者未公开任何潜在的利益冲突。

参考文献

  1. Perret,R.,Sierro,S. R.,Botelho,N. K.,Corgnac,S.,Donda,A.和Romero,P.(2013)。 提高抗原特异性效应物与调节性T细胞比率的佐剂增强肿瘤免疫力。/a> Cancer Res 73(22):6597-6608。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Perret, R., Sierro, S. R., Botelho, N. K., Corgnac, S., Donda, A. and Romero, P. (2014). Vaccine-induced Cytokine Production Detected by Luminex Multiplex Analysis. Bio-protocol 4(16): e1217. DOI: 10.21769/BioProtoc.1217.
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