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Generation and Maturation of Human Monocyte-derived DCs
由人单核细胞诱导树突细胞的产生和成熟   

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参见作者原研究论文

本实验方案简略版
The FASEB Journal
Jan 2014

Abstract

Dendritic cells (DC) are antigen-presenting cells, which play a critical role in the regulation of the adaptive immune response. They act as a bridge between the innate and the adaptive immune systems. An approach to study their function and potentiality is to generate DC-like cells by culturing CD14+ monocyte-enriched peripheral blood mononuclear cells (PBMC). In the presence of GM-CSF and IL-4, these cultures give rise to large numbers of DC-like cells. Generating human-DC from PBMC is a useful tool to study biological functions of human DC.

Keywords: Monocyte-derived dendritic cells (单核细胞来源的树突状细胞), Peripheral blood mononuclear cells (外周血单个核细胞), CD14+ (CD14 +), GM-CSF (GM-CSF), IL-4 (IL-4)

Materials and Reagents

  1. Blood sample to obtain CD14+ cells
  2. Histopaque -1077 (Sigma-Aldrich, catalog number: 10771 )
  3. DPBS (Sigma-Aldrich, catalog number: D8537 )
  4. autoMACS Rinsing Buffer (Miltenyi Biotec, catalog number: 130-091-222 )
  5. Albumin from bovine serum (Sigma-Aldrich, catalog number: A1933 )
  6. FITC-anti-CD14 antibody (BioLegend, catalog number: 301804 )
  7. Recombinant human GM-CSF (Pepro Tech, catalog number: 300-03 )
  8. Recombinant human IL-4 (Pepro Tech, catalog number: 200-04 )
  9. RPMI-1640 medium (Life Technologies, InvitrogenTM, catalog number: 21875034 )
  10. 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
  11. Antibiotics: penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15070 )
  12. Heat inactivated (at 56 °C for 30 min) qualified fetal bovine serum (Life Technologies, InvitrogenTM, catalog number: 26140-087 )
  13. PE-anti-CD1a antibody (BioLegend, catalog number: 300106 )
  14. Recombinant human IL1β (Thermo Fisher Scientific, catalog number: RIL1BI )
  15. Recombinant human TNFα (Thermo Fisher Scientific, catalog number: RTNFAI )
  16. Rabbit Immunoglobulin G (IgG) (Sigma-Aldrich, catalog number: I5006 )
  17. FITC-anti-CD83 antibody (BioLegend, catalog number: 305306 )
  18. PE-anti-CD80 antibody (BioLegend, catalog number: 305208 )
  19. APC-anti-CD40 antibody (BD, catalog number: 555591 )
  20. Isotype-matched mAbs (BioLegend, catalog number: 400113 )
  21. Erylyse buffer (see Recipes)
  22. Complete medium (see Recipes)
  23. AutoMACS running buffer (see Recipes)
  24. FACS wash buffer (see Recipes)
  25. Propidium iodide (Sigma-Aldrich, catalog number: P4170) (see Recipes)

Equipment

  1. Sterilized sierological pipettes (2, 5, 10, 25 ml) (Corning, Costar®, catalog numbers: 4486 , 4487 , 4487 , 4488 , 4489 )
  2. 50 ml conical tubes (BD, Falcon®, catalog number: 352070 )
  3. Human CD14+ magnetic microbeads (Miltenyi Biotec, catalog number: 130-050-201 )
  4. Filtration unit with pore size of 0.22 µm and polyethersulfone (PES) membrane (Thermo Fisher Scientific, catalog number: 431096 )
  5. 6-well tissue culture plate (BD, Falcon®, catalog number: 353046 )
  6. FACS tube (Beckman Coulter, catalog number: 2523749 )
  7. Refrigerated centrifuge with swing out rotor
  8. AutoMACS separator (Miltenyi Biotec)
  9. CyAn ADP flow cytometer (Beckman Coulter)

Procedure

See Notes for a flow chart with the steps to generate DCs.

  1. Take a buffy coat preparation (50 ml) from a healthy donor. Dilute heparinized blood with an equal volume of 1x PBS at room temperature (RT) and put it down slowly over Histopaque to obtain human peripheral blood mononuclear cells (PBMCS).
  2. Add 20 ml of Histopaque (warmed to RT) to 50 ml conical tubes. Then slowly overlay 20 ml of the diluted blood mixture on top of the Histopaque. Centrifuge at 535 x g for 35 min at room temperature with the brake off.
  3. Carefully remove the lymphocyte interface (white ring between the media and Histopaque) with a sterilized serological pipette and transfer to a new 50 ml conical tube (Figure 1).


    Figure 1. A schematic representation of the result of the density gradient stratification (modified by dublinlisacoolsaet.wordpress.com)

  4. Fill the tube with RT PBS to 50 ml and centrifuge at 470 x g for 7 min at room temperature with the brake on (acceleration: 5, deceleration: 5).
  5. Put off the supernatant and re-suspend the cell pellet with 4 ml of Erylyse buffer and incubate at RT for 8 min.
  6. Fill the tube with PBS to 50 ml and centrifuge at 470 x g for 7 min at RT with the brake on.
  7. Resuspend the pellet in 10 ml of PBS, count with dead cell exclusion the PBMCs and centrifuge cell suspension at 470 x g for 10 min.
  8. Isolate human CD14+ cells: Use human CD14+ magnetic microbeads according to the manufacturer’s instructions:
    1. Put off the supernatant and re-suspend the cell pellet in 80 µl of Running Buffer per 2 x 107 total cells in a 50 ml conical tube.
    2. Add 20 µl of CD14 Microbeads per 2 x 107 total cells.
    3. Mix well and incubate for 15 min in the refrigerator (2-8 °C).
    4. Wash cells by adding 1 ml of Running Buffer per 107 cells and centrifuge at 470 x g for 10 min. Put off the supernatant.
    5. Re-suspend up to 108 cells in 500 µl of Running Buffer.
    6. Proceed to magnetic separation with the autoMACS separator.
  9. Collect CD14+ cells, wash with 10 ml complete medium, resuspend the pellet in 10 ml of complete medium and count with dead cell exclusion. CD14+ cells should be 10-15% of PBMC (Figure 2).


    Figure 2. Analysis of CD14+ cells after separation at day 0. A. Histogram: the black peak is the isotype control antibody, the white peak CD14+ cells; B. Dot plot.

  10. Centrifuge at 470 x g for 8 min at room temperature with the brake on and put off the supernatant.
  11. Re-suspend at 5 x 106 cells in 3 ml complete medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4 and then culture cells in 6-well plate. Incubate cells at 37 °C and 5% CO2. (The cytokines are dissolved in DNase/RNase free water and diluted in complete medium.)
  12. Day 3: Added 2 ml/well of fresh complete medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml IL-4.
  13. Day 6: Monocytes are differentiating into immature dendritic cells, they are in suspension and so be careful when removing the plate from incubator. Take medium with cells out of the 6-well plate into a 50 ml tubes and centrifuge at 210 x g for 10 min at 4 °C. Put off the supernatant and re-suspend cells at 1 x 106 cells/ml in complete medium. Collect 1 x 105 cells into FACS tube and add 10 µl PE-anti-CD1a and 10 µl FITC-anti-CD14 antibodies for 20 min at 4 °C and then wash with FACS wash buffer at 470 x g for 5 min. Put off the supernatant and re-suspend cells in 200 µl and run samples on CyAn ADP flow cytometer. The percentage of CD1a+ cells should be higher than 90% as measured by flow cytometer analysis (Figure 3). Immature human derived-DC (iDC) are then ready for experimental use. If iDC population is between 70-90% they can be used for experiments, while if they are less than 70%, restart culture.


    Figure 3. A: expression of CD1a on iDC at day 6; B: expression of CD14 on iDC at day 6

  14. Place 2 x 106 iDCs/well in a 6-well plate and incubate for 24 h in complete medium only or supplemented with 50 ng/ml TNF-α and 50 ng/ml IL-1β to generate mature DC (mDC).
  15. Day 7: Flow cytometry procedure.
    1. Put 6-well plate on ice for 10 min.
    2. Harvest cells with a micropipette with a volume range from 100 µl and 1,000 µl and wash three times with 2 ml of refrigerated PBS. Transfer iDCs and mDCs in two different 15 ml tubes.
    3. Centrifuge at 210 x g for 10 min, and then put off the supernatant.
    4. Re-suspend them in 100 µl of PBS 1x and block non-specific sites with 5 µl rabbit IgG for 5 min at RT.
    5. Divide cells into FACS tube and incubate with 10 µl fluorochrome-conjugated monoclonal antibody and isotype-matched negative controls for 20 min at 4 °C (The following mAbs were used: FITC-anti-CD83, PE-anti-CD80, APC-anti-CD40.).
    6. Filled with 1 ml FACS wash buffer and centrifuge at 1,500 rpm for 5 min and put off the supernatant. Re-suspend cells in 200 µl 1x PBS and analyze samples on CyAn ADP flow cytometer.
    7. Cells were gated according to their light scattering properties to exclude cell debris and contaminating lymphocytes, and no dead cells by propidium iodide positive cells exclusion. (Figure 4).


    Figure 4. A: Dot plot of FS and SS parameter to identify the iDCs; B: gate to exclude the dead cells. We analyzed the cells on gate R1 and gate R3.

Notes

  1. A flow chart with the steps to generate DCs.

Recipes

  1. Erylyse buffer
    500 ml Milliq water
    0.83% NH4Cl
    0.168% Na2CO3
    1 mM EDTA (pH 7.3)
    Sterile filtration
  2. Complete medium
    RPMI medium
    100 U of penicillin
    0.1 mg/ml streptomycin
    50 µM 2-mercaptoethanol
    10% heat-inactivated qualified fetal bovine serum
  3. AutoMACS running buffer
    1 L autoMACS Rinsing buffer
    5 g BSA sterilized by passing through 0.22 μM filter
  4. FACS wash buffer
    1x PBS
    0.2% BSA
    0.1% NaN3
  5. Propidium iodide
    1x PBS
    2% FBS
    0.1% NaN3
    2 mg propidium iodide

Acknowledgments

This protocol was adapted from previous work by Spadaro et al. (2008).

References

  1. Spadaro, M., Caorsi, C., Ceruti, P., Varadhachary, A., Forni, G., Pericle, F. and Giovarelli, M. (2008). Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells. FASEB J 22(8): 2747-2757.
  2. Spadaro, M., Montone, M., Arigoni, M., Cantarella, D., Forni, G., Pericle, F., Pascolo, S., Calogero, R. A. and Cavallo, F. (2014). Recombinant human lactoferrin induces human and mouse dendritic cell maturation via Toll-like receptors 2 and 4. FASEB J 28(1): 416-429.

简介

树突状细胞(DC)是抗原呈递细胞,其在适应性免疫应答的调节中起关键作用。 它们作为先天免疫系统和适应性免疫系统之间的桥梁。 研究它们的功能和潜力的方法是通过培养富集CD14 +单核细胞的外周血单核细胞(PBMC)产生DC样细胞。 在GM-CSF和IL-4存在下,这些培养物产生大量的DC样细胞。 从PBMC产生人类DC是研究人类DC的生物学功能的有用工具。

关键字:单核细胞来源的树突状细胞, 外周血单个核细胞, CD14 +, GM-CSF, IL-4

材料和试剂

  1. 血样获得CD14 + 细胞
  2. Histopaque -1077(Sigma-Aldrich,目录号:10771)
  3. DPBS(Sigma-Aldrich,目录号:D8537)
  4. autoMACS冲洗缓冲液(Miltenyi Biotec,目录号:130-091-222)
  5. 来自牛血清的白蛋白(Sigma-Aldrich,目录号:A1933)
  6. FITC-抗-CD14抗体(BioLegend,目录号:301804)
  7. 重组人GM-CSF(Pepro Tech,目录号:300-03)
  8. 重组人IL-4(Pepro Tech,目录号:200-04)
  9. RPMI-1640培养基(Life Technologies,Invitrogen TM ,目录号:21875034)
  10. 2-巯基乙醇(Life Technologies,Gibco ,目录号:21985-023)
  11. 抗生素:青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15070)
  12. 热灭活(在56℃下30分钟)合格的胎牛血清(Life Technologies,Invitrogen TM,目录号:26140-087)
  13. PE-抗-CD1a抗体(BioLegend,目录号:300106)
  14. 重组人IL1β(Thermo Fisher Scientific,目录号:RIL1BI)
  15. 重组人TNFα(Thermo Fisher Scientific,目录号:RTNFAI)
  16. 兔免疫球蛋白G(IgG)(Sigma-Aldrich,目录号:I5006)
  17. FITC-抗CD83抗体(BioLegend,目录号:305306)
  18. PE-抗CD80抗体(BioLegend,目录号:305208)
  19. APC-anti-CD40抗体(BD,目录号:555591)
  20. 同种型匹配的mAbs(BioLegend,目录号:400113)
  21. Erylyse缓冲区(参见配方)
  22. 完整介质(见配方)
  23. AutoMACS运行缓冲区(参见配方)
  24. FACS洗涤缓冲液(参见配方)
  25. 碘化丙啶(Sigma-Aldrich,目录号:P4170)(参见Recipes)

设备

  1. 灭菌的化疗移液管(2,5,10,25ml)(Corning,Costar ,目录号:4486,4487,4447,4488,4489)
  2. 50ml锥形管(BD,Falcon,目录号:352070)
  3. 人CD14 +磁微珠(Miltenyi Biotec,目录号:130-050-201)
  4. 孔径为0.22μm的过滤单元和聚醚砜(PES)膜(Thermo Fisher Scientific,目录号:431096)
  5. 6孔组织培养板(BD,Falcon ,目录号:353046)
  6. FACS管(Beckman Coulter,目录号:2523749)
  7. 带旋出式转子的冷冻离心机
  8. AutoMACS分离器(Miltenyi Biotec)
  9. CyAn ADP流式细胞仪(Beckman Coulter)

程序

有关生成DC的步骤,请参阅流程图注释。

  1. 从健康供体取血沉棕黄层制剂(50ml)。在室温(RT)下用等体积的1x PBS稀释肝素化的血液,并将其缓慢放在Histopaque上以获得人外周血单核细胞(PBMC S)。
  2. 加入20ml的Histopaque(温热至室温)至50ml锥形管。然后在Histopaque顶部缓慢覆盖20ml稀释的血液混合物。在室温下,在制动关闭的情况下,以535英寸×g离心35分钟。
  3. 用灭菌的血清移液管小心地去除淋巴细胞界面(介质和Histopaque之间的白色环),并转移到新的50ml锥形管中(图1)。


    图1.密度梯度分层(由dublinlisacoolsaet.wordpress.com修改)的结果的示意图

  4. 用RT PBS将管充满至50ml,并在制动(加速度:5,减速度:5)的情况下在室温下以470×g离心7分钟。
  5. 取出上清液并用4ml Erylyse缓冲液重悬细胞沉淀,并在室温下孵育8分钟。
  6. 用PBS填充管至50ml,并在制动开启的情况下在470×g离心7分钟。
  7. 将沉淀重悬在10ml PBS中,用死细胞排除PBMCs并以470×g离心细胞悬液10分钟。
  8. 分离人CD14 + 细胞:根据制造商的说明使用人类CD14 + 磁性微珠:
    1. 取出上清液并将细胞沉淀物在80μl运行缓冲液中每2×10 7个总细胞重悬在50ml锥形管中。
    2. 每2×10 7个总细胞加入20μlCD14微珠
    3. 充分混合并在冰箱(2-8℃)中孵育15分钟
    4. 通过每10 7个细胞加入1ml运行缓冲液洗涤细胞,并在470×g离心10分钟。 倒出上清液。
    5. 在500μl运行缓冲液中重悬最多10个 8 细胞
    6. 使用autoMACS分离器进行磁分离。
  9. 收集CD14 +细胞,用10ml完全培养基洗涤,将沉淀重悬于10ml完全培养基中并用死细胞排除计数。 CD14 + 细胞应为PBMC的10-15%(图2)

    图2.在第0天分离后的CD14 + 细胞的分析。 A。直方图:黑色峰是同种型对照抗体,白色峰CD14 + 细胞; B.点图。

  10. 在室温下以470×g离心8分钟,并打开上清液。
  11. 在补充有100ng/ml GM-CSF和50ng/ml IL-4的3ml完全培养基中以5×10 6个细胞重悬浮,然后在6孔板中培养细胞。在37℃和5%CO 2孵育细胞。 (将细胞因子溶解于不含DNase/RNase的水中,并在完全培养基中稀释)
  12. 第3天:加入2ml /孔补充有100ng/ml GM-CSF和50ng/ml IL-4的新鲜完全培养基。
  13. 第6天:单核细胞分化为未成熟的树突状细胞,它们处于悬浮液中,因此当从培养箱中取出培养板时要小心。取培养基,细胞从6孔板中取出到50ml管中,在4℃下以210×g离心10分钟。取出上清液并以1×10 6个细胞/ml重悬细胞于完全培养基中。收集1×10 5细胞到FACS管中,并在4℃下加入10μlPE-抗-CD1a和10μlFITC-抗-CD14抗体20分钟,然后用FACS洗涤缓冲液在470 xg 5分钟。取出上清液并将细胞重悬在200μl中,并在CyAn ADP流式细胞仪上运行样品。百分比 如通过流式细胞仪分析测量的(图3),CD1a +细胞应该高于90%。然后未成熟的人源DC(iDC)可用于实验用途。如果iDC人口在70-90%之间,他们可以用于实验,而如果他们小于70%,重新启动文化。


    图3.A:在第6天iDC上CD1a的表达; B:第6天CD8在iDC上的表达

  14. 将2×10 6个iDCs /孔置于6孔板中,并在仅含完全培养基或补充有50ng/ml TNF-α和50ng/ml IL-1β的培养基中孵育24小时,以产生成熟DC(mDC)。
  15. 第7天:流式细胞术程序。
    1. 将6孔板在冰上10分钟。
    2. 收获细胞与微量移液器,体积范围从100微升和1000微升,用2毫升冷藏PBS洗涤三次。转移iDC和mDC在两个不同的15毫升管
    3. 在210×g离心10分钟,然后除去上清液
    4. 将它们重新悬浮于100μlPBS 1x中,并在室温下用5μl兔IgG封闭非特异性位点5分钟。
    5. 将细胞分成FACS管,并与10μl荧光染料缀合的单克隆抗体和同种型匹配的阴性对照在4℃孵育20分钟(使用以下mAb:FITC-抗-CD83,PE-抗-CD80,APC-抗 - -CD40。)。
    6. 填充1毫升FACS洗涤缓冲液,并在1,500 rpm离心5分钟,并离开上清液。重悬细胞在200μl1×PBS中,并在CyAn ADP流式细胞仪上分析样品
    7. 根据它们的光散射性质门控细胞以排除细胞碎片和污染淋巴细胞,并且通过碘化丙啶阳性细胞排除没有死亡细胞。 (图4)。


    图4. A:FS和SS参数的点图,用于识别iDC; B:排除死细胞的门。我们分析了门R1和门R3上的细胞

笔记

  1. 具有生成DC的步骤的流程图。

食谱

  1. Erylyse缓冲区
    500 ml Milliq水
    0.83%NH 4 Cl / 0.168%Na 2 CO 3 sub/
    1mM EDTA(pH7.3) 无菌过滤
  2. 完成媒介
    RPMI培养基
    100 U青霉素
    0.1mg/ml链霉素 50μM2-巯基乙醇 10%热灭活的合格胎牛血清
  3. AutoMACS运行缓冲区
    1 L autoMACS冲洗缓冲液
    通过0.22μM过滤器将5g BSA灭菌
  4. FACS洗涤缓冲液
    1x PBS
    0.2%BSA 0.1%NaN 3
  5. 碘化丙啶
    1x PBS
    2%FBS
    0.1%NaN 3
    2mg碘化丙啶

致谢

该协议改编自Spadaro等人之前的工作。(2008)。

参考文献

  1. Spadaro,M.,Caorsi,C.,Ceruti,P.,Varadhachary,A.,Forni,G.,Pericle,F.and Giovarelli,M。(2008)。 乳铁蛋白是先天免疫的主要防御蛋白,是人类树突状细胞的新型成熟因子。/a> FASEB J 22(8):2747-2757。
  2. Spadaro,M.,Montone,M.,Arigoni,M.,Cantarella,D.,Forni,G.,Pericle,F.,Pascolo,S.,Calogero,R.A.和Cavallo, 重组人乳铁蛋白通过Toll样受体2和4诱导人和小鼠树突细胞成熟。 a> FASEB J 28(1):416-429。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Spadaro, M., Montone, M. and Cavallo, F. (2014). Generation and Maturation of Human Monocyte-derived DCs. Bio-protocol 4(15): e1194. DOI: 10.21769/BioProtoc.1194.
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