搜索

Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
采用流式细胞术评估人树突细胞的抗原摄取   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQTM Ovalbumin). The protocol is generated based on data presented in Olivar et al. (2013).

Keywords: Dendritic cells(树突状细胞), Endocytosis(内吞作用), Flow cytometry(流式细胞仪), Fluorescent dye(荧光染料)

Materials and Reagents

  1. Whole blood
  2. RPMI 1640 Medium, GlutaMAXTM (Gibco®, catalog number: 61870 )
  3. DPBS without Ca2+ and Mg2+ (Gibco®, catalog number: 14190-169 )
  4. 100x liquid Penicillin-Streptomycin (Gibco®, catalog number: 15140-122 )
  5. 200 mM L-Glutamine solution (Gibco®, catalog number: 25030-024 )
  6. Fetal Bovine Serum (FBS) (Gibco®, catalog number: 10270106 )
  7. Lipopolysaccharide from Escherichia coli 026:B6 (10 mg) (Sigma-Aldrich, catalog number: L2654 )
  8. Lucifer Yellow CH dilithium salt (25 mg) (Sigma-Aldrich, catalog number: L0259 )
  9. DQTM Ovalbumin (1 mg) (Molecular Probes®, catalog number: D-12053 )
  10. Ficoll-Paque PLUS (General Electric Company, catalog number: 17-1440-03 )
  11. GMP Recombinant Human Interleukin-4 (50 μg, 13 x 106 IU/mg) (Gentaur Molecular Products, catalog number: 04-GMPhuIL4-50 μg )
  12. Recombinant Human GM-CSF (300 μg, 3.88 x 106 IU/vial) (Gentaur Molecular Products, catalog number: 04- RHUGM-CSF-300 μg )
  13. IL-4
  14. Bovine Serum Albumin Fraction V (BSA) (Roche Diagnostics, catalog number: 10735078001 )
  15. FITC-conjugated anti-CD14 (RMO52) (Beckman Coulter, catalog number: IM0645U )
  16. FITC-conjugated anti-IgG2a (7T4-1F5) (Beckman Coulter, catalog number: IM0645U)
  17. Perfect-Count MicrospheresTM (Cytognos S. L., catalog number: CYT-PCM-50 )
  18. NaN3 (Sigma-Aldrich, catalog number: 71289 )
  19. FACS buffer (see Recipes)
  20. Complete medium (see Recipes)
  21. DQ-OVA (1 mg/ml) (see Recipes)
  22. Lucifer Yellow (10 mg/ml) (see Recipes)
  23. rHuIL-4 (500 IU/ml) (see Recipes)
  24. rHuGM-CSF (800 IU/ml) (see Recipes)
  25. LPS (1 mg/ml) (see Recipes)

Equipment

  1. 15 ml Ficoll-Paque PLUS
  2. 60-mm cell culture plates (Corning, catalog number: 15 430166 )
  3. Cytometer tubes (BD Falcon tubes, round-bottom) (Becton, Dickinson and Company, catalog number: 352052 )
  4. Centrifuge Heraeus Multifuge 3 L-R (Heraeus Holding, catalog number: 75004370 )
  5. 37 °C, 5% CO2 cell culture incubator
  6. BD FACSCalibur flow cytometer (Becton, Dickinson and Company, catalog number: 342975 )

Software

  1. CellQuest Pro software (Becton, Dickinson and Company, catalog number: 643436 )

Procedure

  1. Dilute 25 ml of buffy coat (initial leukocyte concentrate from a whole blood donation) with the same volume of DPBS.
  2. Prepare two 50 ml tubes with 15 ml Ficoll-Paque PLUS. Carefully layer 25 ml of the diluted blood sample on Ficoll-Paque PLUS. Important: when layering the sample do not mix Ficoll-Paque PLUS and the diluted blood sample.
  3. Centrifuge at 400 x g for 25 min at 18-20 °C. Important: brakes off.
  4. Soak up the white interphase between the diluted plasma fraction and the transparent ficoll fraction with a pipette and transfer it into a fresh tube.
  5. Wash twice with DPBS.
  6. Resuspend the pellet in 5 ml DPBS.
  7. In a cytometer tube mix 3 μl of FITC-conjugated anti-CD14 antibody plus 60 μl DPBS and 20 μl of cellular suspension.
  8. Incubate 15-18 min at room temperature.
  9. Add 120 μl DPBS and count the number of CD14+ monocytes by flow cytometry using Perfect-Count MicrospheresTM according to the manufacturer’s instructions.
  10. Plate monocytes at 1 x 106 cells/ml in 60-mm culture plates, in RPMI 1640 medium without serum, and allow to adhere for 2 h at 37 °C in 5% CO2.
  11. Remove the non-adherent cells by washing in DPBS. The final population of adherent cells contains 75-80% of monocytes, as demonstrated by flow cytometry of anti-CD14–stained isolates.
  12. Generate monocyte-derived DCs by supplementing the monocyte cultures with 1 ml of complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml).
  13. At day 3 add 1ml of complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml).
  14. For DC maturation, at day 5 replace the old medium with fresh complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml) and stimulate the immature DCs for 48 h with 5 μg/ml LPS.
  15. Harvest the non-adherent cells floating in the culture medium in a 15 ml tube (at day 5 for immature DCs; at day 7 for mature DCs). Add 2 ml DBPS (37 °C), rinse and collect the adhered cells by pipetting. Wash twice more with DPBS and pool both floating and adherent cells. Centrifugue and resuspend the pellet in 500 μl of complete medium.
  16. Prepare two cytometer tubes with 60 μl of complete medium plus 4 μl DQ-OVA (stock: 1 mg/ml) at 37 °C or 0 °C.
  17. Prepare two cytometer tubes with 60 μl of complete medium plus 6 μl Lucifer Yellow CH (stock: 10 mg/ml) at 37 °C or 0 °C.
  18. Add 100 μl of cell suspension (~ 2 x 105 cells/ml) to each cytometer tube.
  19. Incubation time: 15 min for DQ-OVA; 120 min for Lucifer Yellow CH. The fluorescence of OVA labeled with BODIPY FL dye (DQ-OVA) is self-quenched until the OVA is taken up via the mannose receptor and degraded only by endolysosomal proteases. Lucifer Yellow CH (LY) is a hydrophilic tracer for fluid-phase macropinocytosis. LY is not degraded and is nontoxic at concentrations up to 6 mg/ml.
  20. Stop the incubations by adding 1 ml cold FACS buffer.
  21. Wash the cells two times with cold FACS buffer.
  22. Analyze the incorporated fluorescence of both immature DCs (Figure 1) and mature DCs using flow cytometry. Compare the histograms and corresponding mean fluorescence intensities (MFI) between cells incubated at 37 °C (specific uptake) and cells incubated at 0 °C (non-specific uptake: passive diffusion,…).


    Figure 1. Analysis of the endocytic activity of immature DCs by flow cytometry. The endocytic activity of monocyte-derived immature DCs was assessed measuring the uptake of the fluorescent reporters DQ-OVA (receptor-mediated endocytosis) and Lucifer Yellow CH (fluid-phase endocytosis). Representative histograms are shown. Dye uptake controls are displayed in gray. The median fluorescence intensities (MFI) for the different fluorescent cell populations are indicated in each histogram.

Recipes

  1. FACS buffer (500 ml)
    Mix 5 g BSA and 0.5 g NaN3 with 500 ml 1x DPBS
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  2. Complete medium
    RPMI 1640 medium, GlutaMAXTM
    100 μg/ml streptomycin
    100 IU/ml penicillin
    2 mM L-glutamine
    10% heat-inactivated FBS
    GM-CSF 800 IU/ml
    IL-4 500 IU/ml
    Stored at 4 °C
  3. DQ-OVA (1 mg/ml)
    A 1 mg/ml solution can be prepared by dissolving the contents of one vial in 1 ml of DPBS. Once reconstituted, the solution should be stored at -20 °C, protected from light.
  4. Lucifer Yellow (10 mg/ml)
    A 10 mg/ml solution can be prepared by dissolving the contents of one vial in 2.5 ml of dH2O. Once reconstituted, the solution should be stored at 4 °C, protected from light.
  5. rHuIL-4 (500 IU/ml)
    A 500 IU/ml solution can be prepared by dissolving the contents of one vial in 500 μl of dH2O. Once reconstituted, the solution should be stored at -80 °C.
  6. rHuGM-CSF (800 IU/ml)
    A 800 IU/ml solution can be prepared by dissolving the contents of one vial in 2 ml of dH2O. Once reconstituted, the solution should be stored at -80 °C.
  7. LPS (1 mg/ml)
    A 1 mg/ml solution can be prepared by dissolving the contents of one vial in 1 ml of DPBS. Once reconstituted, the solution should be stored at -20 °C.

Acknowledgments

This protocol was adapted from the previously published study, Olivar et al. (2013), and was supported by the Ministerio de Ciencia e Innovación (Madrid, Spain), through grant PI10/1073 from the “Fondo de Investigaciones Sanitarias” (FIS-ISCIII), and from 2009SGR1490 (Generalitat de Catalunya) to JMA. JMA is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES06/012).

References

  1. Olivar, R., Luque, A., Naranjo-Gomez, M., Quer, J., Garcia de Frutos, P., Borras, F. E., Rodriguez de Cordoba, S., Blom, A. M. and Aran, J. M. (2013). The α7β0 isoform of the complement regulator C4b-binding protein induces a semimature, anti-inflammatory state in dendritic cells. J Immunol 190(6): 2857-2872.

简介

树突状细胞的抗原摄取是诱导抗原特异性T细胞应答的第一个关键步骤。 这种基于流式细胞术的方案描述了通过两种不同的机制分析树突状细胞摄取可溶性抗原:非特异性大细胞增多症(使用Lucifer Yelloy CH)和受体介导的内吞作用(使用DQ TM 卵清蛋白) 。 该方案是基于Olivar等人(2013)中提供的数据产生的。

关键字:树突状细胞, 内吞作用, 流式细胞仪, 荧光染料

材料和试剂

  1. 全血
  2. RPMI 1640 Medium,GlutaMAX TM (Gibco ,目录号:61870)
  3. DPBS,不含Ca 2+ 2+和Mg 2+ 2+ (Gibco ,目录号:14190-169)。
  4. 100x液体青霉素 - 链霉素(Gibco ,目录号:15140-122)
  5. 200mM L-谷氨酰胺溶液(Gibco ,目录号:25030-024)
  6. 胎牛血清(FBS)(Gibco ,目录号:10270106)
  7. 来自大肠杆菌O26:B6(10mg)(Sigma-Aldrich,目录号:L2654)的脂多糖
  8. 萤光黄黄色CH二锂盐(25mg)(Sigma-Aldrich,目录号:L0259)
  9. DQ缓冲蛋白(1mg)(Molecular Probes ,目录号:D-12053)
  10. Ficoll-Paque PLUS(General Electric Company,目录号:17-1440-03)
  11. GMP重组人白介素-4(50μg,13×10 6 IU/mg)(Gentaur Molecular Products,目录号:04-GMPhuIL4-50μg)
  12. 将重组人GM-CSF(300μg,3.88×10 6个IU /瓶)(Gentaur Molecular Products,目录号:04-RHUGM-CSF-300μg)
  13. IL-4
  14. 牛血清白蛋白馏分V(BSA)(Roche Diagnostics,目录号:10735078001)
  15. FITC缀合的抗CD14(RMO52)(Beckman Coulter,目录号:IM0645U)
  16. FITC缀合的抗IgG2a(7T4-1F5)(Beckman Coulter,目录号:IM0645U)
  17. Perfect-Count Microspheres TM (Cytognos S.L.,目录号:CYT-PCM-50)
  18. NaN 3(Sigma-Aldrich,目录号:71289)
  19. FACS缓冲区(参见配方)
  20. 完整介质(见配方)
  21. DQ-OVA(1mg/ml)(参见Recipes)
  22. 萤光黄(10mg/ml)(见配方)
  23. rHuIL-4(500IU/ml)(参见配方)
  24. rHuGM-CSF(800IU/ml)(参见配方)
  25. LPS(1mg/ml)(参见配方)

设备

  1. 15 ml Ficoll-Paque PLUS
  2. 60-mm细胞培养板(Corning,目录号:15430166)
  3. 细胞计数管(BD Falcon管,圆底)(Becton,Dickinson and Company,目录号:352052)
  4. 离心机Heraeus Multifuge 3 L-R(Heraeus Holding,目录号:75004370)
  5. 37℃,5%CO 2细胞培养箱中培养
  6. BD FACSCalibur流式细胞仪(Becton,Dickinson and Company,目录号:342975)

软件

  1. CellQuest Pro软件(Becton,Dickinson and Company,目录号:643436)

程序

  1. 用相同体积的DPBS稀释25ml血沉棕黄层(来自全血捐献的初始白细胞浓缩物)。
  2. 准备两个50毫升管用15毫升Ficoll-Paque PLUS。 小心地将25ml稀释的血液样品在Ficoll-Paque PLUS上。 重要:当分层样品不要混合Ficoll-Paque PLUS和稀释的血液样品
  3. 在18-20℃下以400×g离心25分钟。 重要:刹车。
  4. 用移液管吸取稀释的血浆级分和透明的ficoll级分之间的白色界面,并将其转移到新管中。
  5. 用DPBS洗涤两次。
  6. 将沉淀重悬在5ml DPBS中
  7. 在细胞计量管中混合3μlFITC缀合的抗CD14抗体加上60μlDPBS和20μl细胞悬浮液。
  8. 在室温下孵育15-18分钟。
  9. 加入120μlDPBS,并通过流式细胞术使用Perfect-Count Microspheres TM 根据制造商的说明计数CD14 +单核细胞的数目。
  10. 在60-mm培养板中,在不含血清的RPMI 1640培养基中,以1×10 6个细胞/ml将单核细胞平板,并允许在37℃下在5%CO 2中粘附2小时 。
  11. 通过在DPBS中洗涤去除非粘附细胞。 最终的粘附细胞群包含75-80%的单核细胞,如通过抗CD14染色的分离株的流式细胞术所证明的。
  12. 通过用1ml完全RPMI 1640培养基加上GM-CSF(800IU/ml)和IL-4(500IU/ml)补充单核细胞培养物产生单核细胞衍生的DC。
  13. 在第3天加入1ml完全RPMI 1640培养基加上GM-CSF(800IU/ml)和IL-4(500IU/ml)。
  14. 对于DC成熟,在第5天用新鲜的完全RPMI 1640培养基加上GM-CSF(800IU/ml)和IL-4(500IU/ml)替换旧培养基,并用5μg/ml刺激未成熟DC 48小时LPS。
  15. 收获漂浮在培养基中的非粘附细胞在15毫升管(在第5天为未成熟的DC;在第7天为成熟的DC)。加入2ml DBPS(37℃),冲洗并通过吸移收集粘附的细胞。用DPBS洗涤两次以上,并漂浮和贴壁细胞。离心并将沉淀重悬于500μl完全培养基中
  16. 准备两个细胞计管与60微升的完整培养基加4微升DQ-OVA(股票:1毫克/毫升)在37℃或0℃。
  17. 在37°C或0°C下用60μl完全培养基加6μlLucifer Yellow CH(原液:10 mg/ml)制备两个细胞计数管。
  18. 向每个细胞计数管中加入100μl细胞悬浮液(〜2×10 5个细胞/ml)。
  19. 保温时间:DQ-OVA为15分钟; 120分钟Lucifer黄色CH。用BODIPY FL染料(DQ-OVA)标记的OVA的荧光自淬灭,直到OVA通过甘露糖受体摄取并仅被内溶酶体蛋白酶降解。 Lucifer Yellow CH(LY)是一种亲水示踪剂,用于流体相巨噬细胞增多。 LY不降解,并且在高达6mg/ml的浓度下是无毒的。
  20. 通过加入1ml冷FACS缓冲液停止孵育
  21. 用冷的FACS缓冲液洗涤细胞两次
  22. 使用流式细胞术分析未成熟DC(图1)和成熟DC的掺入的荧光。比较在37℃(特异性摄取)和在0℃孵育的细胞(非特异性摄取:被动扩散,...)下孵育的细胞的直方图和相应的平均荧光强度(MFI)。


    图1.通过流式细胞术分析未成熟DC的内吞活性。 评估单核细胞衍生的未成熟DC的内吞活性,测量荧光报告子DQ-OVA(受体介导的内吞作用)和萤光黄黄色CH(流体相内吞作用)的摄取。显示了代表性直方图。染料摄取对照显示为灰色。在每个直方图中指示不同荧光细胞群的中值荧光强度(MFI)。

食谱

  1. FACS缓冲液(500ml) 将5g BSA和0.5g NaN 3与500ml 1x DPBS混合 过滤灭菌(0.45μm)
    储存在4°C
  2. 完成媒介
    RPMI 1640培养基,GlutaMAX TM
    100μg/ml链霉素 100 IU/ml青霉素
    2mM L-谷氨酰胺 10%热灭活的FBS GM-CSF 800 IU/ml
    IL-4 500 IU/ml
    储存在4°C
  3. DQ-OVA(1mg/ml) 1mg/ml溶液可以通过将一个小瓶的内容物溶解在1ml DPBS中来制备。 一旦重建,溶液应储存在-20°C,避光
  4. 萤光黄(10mg/ml) 10mg/ml溶液可以通过将一个小瓶的内容物溶解在2.5ml dH 2 O中制备。 重建后,溶液应储存在4°C,避光保存
  5. rHuIL-4(500IU/ml) 可以通过将一个小瓶的内容物溶解在500μldH 2 O中制备500IU/ml溶液。 一旦重建,溶液应储存在-80℃
  6. rHuGM-CSF(800IU/ml) 可以通过将一个小瓶的内容物溶解在2ml dH 2 O中制备800IU/ml溶液。 一旦重建,溶液应储存在-80℃
  7. LPS(1mg/ml)
    1mg/ml溶液可以通过将一个小瓶的内容物溶解在1ml DPBS中来制备。 重构后,溶液应储存在-20°C

致谢

该方案改编自先前公开的研究,Olivar等人(2013),并且由西班牙马德里部长办公室(Madrid,Spain)支持,通过授予PI10/1073从"Fondo de Investigaciones Sanitarias"(FIS-ISCIII),以及从2009SGR1490(Generalitat de Catalunya)到JMA。 JMA由SNS-Dpt的"研究者合并计划"赞助。 Salut Generalitat de Catalunya(Exp。CES06/012)。

参考文献

  1. Olivar,R.,Luque,A.,Naranjo-Gomez,M.,Quer,J.,Garcia de Frutos,P.,Borras,FE,Rodriguez de Cordoba,S.,Blom,AM and Aran,JM 。 补体调节剂C4b结合蛋白的α7βO同种型诱导树突状细胞中的半衰期,抗炎状态 细胞。 190(6):2857-2872。
  • 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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luque, A., Cárdenas-Brito, S., Olivar, R. and Aran, J. M. (2013). Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry. Bio-protocol 3(22): e974. DOI: 10.21769/BioProtoc.974.
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。