搜索

Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting
采用流式细胞术分离脾脏中树突细胞   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The spleen is a vastly vasculated organ and consists of a complex organized network of innate and adaptive immune cells. This permits the specialized functions of the spleen such as antibacterial and antifungal immunity and iron metabolism among others (Mebius and Kraal, 2005). Different dendritic cell (DC) subsets reside in the spleen and can be defined by the expression of unique surface markers. These DC subsets are recognized to perform non-redundant functions in the immune system (Merad et al., 2013). In our recent study, we found that Inositol Requiring Enzyme (IRE)-1 is specifically activated in splenic CD8a+ DCs. Furthermore, loss of X-box binding protein (XBP)-1 – the transcription factor regulated by IRE-1 – resulted in defective cross-presentation of dead cell associated antigens by splenic CD8a+ DCs (Osorio et al., 2014). This protocol allows the isolation of specific DC subsets for experimental use ex-vivo.

Materials and Reagents

  1. Mice (Jackson Laboratories, C57Bl/6 )
  2. 2,2,2-tribromoethanol (Sigma-Aldrich, catalog number: T48402 )
  3. 2-methyl-2-butanol (Sigma-Aldrich, catalog number: 240486 )
  4. PBS (Gibco, catalog number: 10010-015 )
  5. 0.5 M EDTA (Lonza, catalog number: 51234 )
  6. 2-mercaptoethanol (Sigma-Aldrich, catalog number M3148 )
  7. BSA (Amresco, catalog number: 0332-500g )
  8. HBSS (Life Technologies, catalog number: 24020-091 )
  9. RPMI 1640 GlutaMax (Life Technologies, catalog number: 61870-010 )
  10. Gentamycin (Gibco, catalog number: 15710-049 )
  11. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  12. Liberase TM (Roche Diagnostics, catalog number: 0 5401127001 ) (dilute 50 mg in 50 ml of RPMI, aliquot and stored at -20 °C)
  13. DNase I (Roche, catalog number: 0 4536282001 ) (dilute content in 1 ml PBS, aliquot and stored at -20 °C)
  14. FACS antibodies
    1. CD3 - FITC (eBioscience, clone 145-2C11, dilution 1/300)
    2. CD19 - FITC (eBioscience, clone 1D3, dilution 1/500)
    3. CD11c - PE-eFluor610 (eBioscience, N418, dilution 1/300)
    4. MHCII - APC-Cy7 (Biolegend, clone M5/114.15.2, dilution 1/1,000)
    5. CD8a - PE-Cy5 (BD-Pharmingen, clone 53-6.7, dilution 1/600)
    6. CD11b - PE-Cy7 (BD-Pharmingen, clone M1/70, dilution 1/800)
    7. CD64 - AF647 (BD-Pharmingen, clone X54-5/7.1, dilution 1/100)
    8. CD16/CD32 (Produced in-house, clone 2.4G2, dilution 1/200)
      Note: The use of 2.4G2 minimizes background fluorescence by inhibiting non-specific binding of antibodies to FcG – receptors present on immune cells.
  15. Fixable viability dye eFluor 506 (eBioscience, catalog number: 65-0866-18 )
    1. Reconstitute vial with 400 μl PBS
    2. Aliquot and store vials at -80 °C
    3. Use at a dilution of 1/200
  16. Anti-FITC microbeads (Miltenyi, catalog number 130-048-701 )
  17. LD columns (Miltenyi, catalog number: 130-042-901 )
  18. Ultra comp eBeads (eBioscience, catalog number: 01-2222-42 )
  19. Trypan blue (Sigma-Aldrich, catalog number: T8154 )
  20. Avertin (see Recipes)
  21. 2x digestion medium (see Recipes)
  22. MACS buffer (see Recipes)
  23. R10 medium (see Recipes)
  24. Osmotic lysis buffer (see Recipes)

Equipment

  1. 100 μm Nylon cell strainer (Falcon, catalog number: 352360 )
  2. 15 ml tubes (Falcon, catalog number: 430791 )
  3. 35 mm Petri Dishes
  4. 5ml syringe (Norm-Ject, catalog number: 4020-000V0 )
  5. 25 Gauge needle (Terumo, catalog number: NN-2516R )
  6. Pasteur pipette
  7. Forceps
  8. Surgical scissor/scalpel
  9. 37 °C warm water bath
  10. Shaker
  11. Centrifuge
  12. Aspiration device
  13. MACS MultiStand (Miltenyi, catalog number: 130-042-303 )
  14. Midi MACS (Miltenyi, catalog number: 130-042-302 )
  15. Polystyrene 5 ml tubes with 100 μm cell strainer cap (BD Falcon, catalog number: 352235 )
  16. Polypropylene 5ml tubes (BD Falcon, catalog number: 352063 )
  17. Microscope
  18. Bürker-Türk counting plate
  19. Multicolour FACS sorter (BD Aria II or equivalent cell sorter equipped with 405 nm, 488 nm and 633 nm lasers and appropriate filterset)

Procedure

  1. Euthanize the mice by injecting 0.5 ml of avertin intraperitoneally with a 25 Gauge needle. The mice become unresponsive after 1-2 min.
    Note: Verify the institution approved ethical regulations on animal welfare if other euthanization methods are in place.
  2. Check the absence of cerebrospinal reflexes by pinching the paw of the posterior limbs with a forceps.
  3. After spraying the animal with 70% ethanol, open the right flank and remove the spleen. Remove any fatty tissue that is connected with the spleen and place the organ in 15 ml tube with 5 ml of ice-cold HBSS.
  4. Bring the spleen to a 35 mm Petri dish and mince the organ into pieces by using the scissors. To obtain a good dissociation of the tissue, proceed until no pieces are visible with the bare eyes.
  5. Add 1 ml of ice-cold RPMI, bring the cell suspension to a 15 ml Falcon tube and put on ice. Complete all other organs before adding the digestion medium.
  6. Add 1 ml of 37 °C preheated 2x digestion medium to the cell suspension and bring all organs to the 37 °C warm water bath. Make sure the shaker is put to its maximum setting.
    Note: Shaking the sample will allow more efficient digestion of the tissue by decreasing its precipitation.
  7. Incubate the cell suspension for 15 min and dissociate the cell suspension by pipetting the solution up and down through a Pasteur pipette.
  8. Repeat step 7 once more.
  9. Add 10 ml of ice-cold MACS buffer and spin down cell suspension at 400 x g 7 min 4 °C.
  10. Aspirate the supernatant and resuspend the cell pellet in 2 ml of ice-cold osmotic lysis buffer. Incubate for 4’ at room temperature and subsequently add 10 ml of ice-cold MACS buffer. Spin down cell suspension at 400 x g 7 min 4 °C.
  11. Resuspend cells in 10 ml of ice-cold MACS buffer and filter through a 100 μm cell strainer. One can reuse the 15 ml tube to collect the filtered cells. Take a 20 μl counting sample and spin down rest of cell suspension at 400 x g 7 min 4 °C.
  12. Add 180 μl of trypan blue to the 20 μl of counting sample and mix well. Count cells. After digestion, 1 spleen will normally contain up to 60-100 x106 cells.
  13. Remove supernatant from cell pellet and resuspend in antibody mix I at a concentration of 50 x 106 cells per 1 ml of mix (see table 1). Stain 20 min on ice in the dark. Add 10 ml of ice-cold MACS buffer and spin down 7 min 400 x g 4 °C.

    Table 1. Antibody mix I in MACS buffer
    Label
    Marker
    Clone
    Dilution
    FITC
    CD3
    145-2C11
    1/300
    FITC
    CD19
    1D3
    1/500
    -
    CD16/CD32
    2.4G2
    1/200

  14. Remove supernatant from cell pellet and add 90 μl of ice-cold MACS buffer per 10 x 106 cells.
  15. Add 5-10 μl/10 x 106 cells of anti-FITC microbeads. Resuspend and incubate 15 min on ice. Add 10 ml of ice-cold MACS buffer and spin down 7 min 400 x g 4 °C. Discard supernatant.
  16. Prepare LD columns according to manufacturer’s protocol.
  17. After preparation, place a 15 ml tube on ice under the LD column.
  18. Resuspend cell pellet in 1 ml of ice-cold MACS buffer and filter through a 100 μm cell strainer on the LD column.
    Note: This minimizes the probability of cell clots obstructing the LD column. After the 1 ml of cell suspension has run through, wash 2 times with 2 ml of ice-cold MACS buffer. Make sure that cell suspension runs through each time. Discard the column and spin down the effluent (7 min 400 x g 4 °C). Aspirate the supernatant.
  19. Stain cell pellet in 500 μl of antibody mix II. Stain 20 min on ice in the dark. Add 10 ml of ice-cold MACS buffer and spin down 7 min 400 x g 4 °C. Aspirate supernatant and resuspend in 1 ml of ice-cold MACS buffer. Bring the cell suspension to a 5 ml polystyrene tube with a cell strainer cap to remove possible clots.

    Table 2. Antibody mix II in PBS
    Label
    Marker
    Clone
    Dilution
    FITC
    CD3
    145-2C11
    1/300
    FITC
    CD19
    1D3
    1/500
    PE-eFluor610
    CD11c
    N418
    1/300
    PE-Cy5
    CD8a
    53-6.7
    1/600
    PE-Cy7
    CD11b
    M1/70
    1/800
    AF-647
    CD64
    X54-5/7.1
    1/100
    APC-Cy7
    MHCII
    M5/114.15.2
    1/1,000
    eFluor
    506
    Viability
    1/200
    -
    CD16/CD32
    2.4G2
    1/200
    Note: Use of PBS is necessary for optimal staining of dead cells with the Fixable viability dye eFluor 506.

  20. Single stains are prepared by diluting 1 drop of Ultra comp eBeads in 2 ml of PBS. Make sure to shake the beads thoroughly before use as precipitation occurs. Bring 200 μl of this bead suspension to seven 5 ml reaction tubes (6 fluorochromes + 1 unstained sample). Antibodies conjugated to bright fluorochomes (PE-eFluor610, Pe-Cy5, AF-647) are diluted 1/2,000, other antibodies are diluted 1/400. Stain 15 min on ice in the dark. Add 1 ml of PBS and spin down 7 min 400 x g 4 °C. Remove supernatant and resuspend beads in 200 μl of PBS. Proceed to the cell sorter.
    Note: Viability dyes do not bind to the Ultra comp eBeads. Since we exclude dead cells (defined by a positive staining for eFluor 506) there is no need to compensate this fluorochrome. If compensation is preferred, cells can be stained with a 1 in 200 dilution of the eFluor 506 viability dye in PBS. Stain 20 min on ice in the dark. Wash cells with 1ml of PBS and spin down 7 min 400 x g 4 °C. Resuspend the cells in 200 μl of PBS.
  21. An example of a gating setup is shown below.


    Figure 1. Gating strategy of splenic DCs. A strict gating strategy in the FSC-H/FSC-A plot allows the exclusion of doublets. The CD11b+ DCs (Lin- CD11c+ MHCII+ CD11b+ cells) contain a fraction of monocyte derived cells (specific infections with pathogens or inflammation can drastically increase this cell population, contaminating the CD11b+ DC gate). The conventional CD11b+ DCs can be identified by exclusion of the CD64+ cells.

Notes

  1. One should take in mind to work as fast as possible. Once removed from the live animal splenic DC subsets viability is quickly compromised. Furthermore, working with cold reagents improves overall survival.
  2. A good dissociation of the tissue by use of scissor and Pasteur pipettes and adequate incubation of the splenocytes in the digestion medium is crucial for optimal cell retrieval. When adjusting the antibody mix, keep in mind that the enzymatic digestion can potentially cleave off surface proteins expressed on the cell types of interest.
  3. In optimal conditions, negative selection with the use of MACS beads removes 90-95% of CD3e+ and CD19+ cells. This reduces the number of cells to approximate 5-10 x 106 cells and a total sorting time to approximate 20 min.
  4. One can sort 80,000-120,000 CD8a+ DCs and 400,000-600,000 CD11b+ DCs from 1 spleen (in homeostatic conditions). The CD11b+ DCs can be further subdivided by the use of surface markers ESAM or CD4.
  5. In the case that the cells are to be cultured, the scientist should keep in mind to work sterile from the moment the spleen is obtained (step 4). Prior to use, the buffers can be filtered to remove potential pathogens using a 0.2 μm filter.
  6. Cells can be brought into culture in R10 medium with the addition of 250 ng/ml of recombinant murine Flt3L. Without any further stimulation; survival of these dendritic cell subsets is less than 60% after 24 h of culture.

Recipes

  1. Avertin (for 40 ml)
    Dissolve 1 g of 2,2,2-tribromoethanol in 1 ml of 2-methyl-2-butanol
    Resuspend in 39 ml of PBS and shake over night at room temperature
    Avoid direct exposure to light and store at 4 °C
  2. 2x digestion medium
    Reconstitute RPMI 1640 with:
    1/500 DNAse I (10 IU/ml)
    1/25 liberase TM (0.42 Wünsch Unit/ml)
  3. MACS buffer
    1x PBS
    2% FCS (or 2% BSA)
    5 mM EDTA
    Note: This minimizes adherence of cells to plastics by chelating Ca2+.
    Filter sterilize over a 0.2 μm filter and store at 4 °C
  4. R10 medium
    Reconstitute RPMI1640-Glutamax with:
    10% FBS
    50 μg/ml gentamycin
    50 μM beta-mercaptoethanol
  5. Osmotic lysis buffer (500 ml total)
    450 ml MilliQ water (or other source of ultra-pure ddH2O)
    4.145 g ammonium chloride
    100 μl 500 mM EDTA
    Adjust pH to 7.1-7.4
    Add MilliQ water up to 500 ml
    Filter sterilize over a 0.2 μm filter and stored at 4 °C

Acknowledgments

This work was supported by the European Research Council, the European Union Seventh Framework Programme, the Fonds Wetenschappelijk Onderzoek Vlaanderen program (FWO) and the Agentschap voor Innovatie door Wetenschap en Technologie (IWT).

References

  1. Merad, M., Sathe, P., Helft, J., Miller, J. and Mortha, A. (2013). The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31: 563-604.
  2. Mebius, R. E. and Kraal, G. (2005). Structure and function of the spleen. Nat Rev Immunol 5(8): 606-616.
  3. Osorio, F., Tavernier, S. J., Hoffmann, E., Saeys, Y., Martens, L., Vetters, J., Delrue, I., De Rycke, R., Parthoens, E., Pouliot, P., Iwawaki, T., Janssens, S. and Lambrecht, B. N. (2014). The unfolded-protein-response sensor IRE-1alpha regulates the function of CD8alpha+ dendritic cells. Nat Immunol 15(3): 248-257.

简介

脾脏是一个巨大的血管的器官,由一个复杂的有组织的网络的先天和适应性免疫细胞组成。 这允许脾的特殊功能,例如抗菌和抗真菌免疫和铁代谢等(Mebius和Kraal,2005)。 不同的树突状细胞(DC)子集驻留在脾脏中,并且可以通过表达独特的表面标志物来定义。 认识到这些DC亚类在免疫系统中执行非冗余功能(Merad等人,2013)。 在我们最近的研究中,我们发现肌醇需求酶(IRE)-1在脾CD8a + DC中特异性激活。 此外,X-box结合蛋白(XBP)-1(由IRE-1调节的转录因子)的缺失导致脾CD8a + DC(Osorio em> et al。,2014)。 该协议允许分离用于实验使用的特异性DC亚群ex-vivo 。

材料和试剂

  1. 小鼠(Jackson Laboratories,C57Bl/6)
  2. 2,2,2-三溴乙醇(Sigma-Aldrich,目录号:T48402)
  3. 2-甲基-2-丁醇(Sigma-Aldrich,目录号:240486)
  4. PBS(Gibco,目录号:10010-015)
  5. 0.5 M EDTA(Lonza,目录号:51234)
  6. 2-巯基乙醇(Sigma-Aldrich,目录号M3148)
  7. BSA(Amresco,目录号:0332-500g)
  8. HBSS(Life Technologies,目录号:24020-091)
  9. RPMI 1640 GlutaMax(Life Technologies,目录号:61870-010)
  10. 庆大霉素(Gibco,目录号:15710-049)
  11. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
  12. Liberase TM(Roche Diagnostics,目录号:05401127001)(在50ml RPMI中稀释50mg,等分并保存在-20℃)
  13. DNase I(Roche,目录号:04536282001)(在1ml PBS中稀释的内含物,等分并保存在-20℃)
  14. FACS抗体
    1. CD3-FITC(eBioscience,克隆145-2C11,稀释1/300)
    2. CD19-FITC(eBioscience,克隆1D3,稀释1/500)
    3. CD11c-PE-eFluor610(eBioscience,N418,稀释1/300)
    4. MHCII-APC-Cy7(Biolegend,克隆M5/114.15.2,稀释1/1000)
    5. CD8a-PE-Cy5(BD-Pharmingen,克隆53-6.7,稀释度1/600)
    6. CD11b-PE-Cy7(BD-Pharmingen,克隆M1/70,稀释1/800)
    7. CD64-AF647(BD-Pharmingen,克隆X54-5/7.1,稀释1/100)
    8. CD16/CD32(内部生产,克隆2.4G2,稀释1/200)
      注意:使用2.4G2通过抑制使背景荧光最小化 抗体对存在于免疫上的FcG-受体的非特异性结合 细胞。
  15. 可固定活性染料eFluor 506(eBioscience,目录号:65-0866-18)
    1. 用400μlPBS重构小瓶
    2. 将样品瓶和存储瓶置于-80°C
    3. 稀释度为1/200时使用
  16. 抗FITC微珠(Miltenyi,目录号130-048-701)
  17. LD柱(Miltenyi,目录号:130-042-901)
  18. Ultra Comp eBeads(eBioscience,目录号:01-2222-42)
  19. 台盼蓝(Sigma-Aldrich,目录号:T8154)
  20. Avertin(参见食谱)
  21. 2x消化培养基(参见配方)
  22. MACS缓冲区(参见配方)
  23. R10介质(见配方)
  24. 渗透裂解缓冲液(参见配方)

设备

  1. 100μm尼龙细胞过滤器(Falcon,目录号:352360)
  2. 15ml管(Falcon,目录号:430791)
  3. 35 mm培养皿
  4. 5ml注射器(Norm-Ject,目录号:4020-000V0)
  5. 25号针(Terumo,目录号:NN-2516R)
  6. 巴斯德移液器
  7. 镊子
  8. 外科剪刀/手术刀
  9. 37°C温水浴
  10. 振动器
  11. 离心机
  12. 抽吸装置
  13. MACS MultiStand(Miltenyi,目录号:130-042-303)
  14. Midi MACS(Miltenyi,目录号:130-042-302)
  15. 具有100μm细胞过滤帽的聚苯乙烯5ml管(BD Falcon,目录号:352235)
  16. 聚丙烯5ml管(BD Falcon,目录号:352063)
  17. 显微镜
  18. Bürker-Türk计数板
  19. 多色FACS分选机(BD Aria II或配备405 nm,488 nm和633 nm激光器及相应滤光片组的等效细胞分选机)

程序

  1. 安乐死小鼠腹腔注射0.5毫升的avertin用25号针。 小鼠在1-2分钟后变得无反应。
    注意:如果其他安乐死方法到位,请验证机构批准的动物福利道德规范。
  2. 通过用镊子夹住后肢的爪子来检查脑脊髓反射的缺失。
  3. 用70%乙醇喷洒动物后,打开右胁腹并取出脾脏。取出与脾相连的任何脂肪组织,并将器官置于含有5ml冰冷HBSS的15ml管中。
  4. 将脾脏置于35mm培养皿中,使用剪刀将器官切碎。为了获得良好的组织离解,继续直到用裸眼看不到任何碎片
  5. 加入1ml冰冷的RPMI,将细胞悬浮液置于15ml Falcon管中并置于冰上。在添加消化培养基之前完成所有其他器官。
  6. 向细胞悬浮液中加入1ml 37℃预热的2x消化培养基,并将所有器官置于37℃温水浴中。确保振动筛处于最大设置。
    注意:摇动样品会减少其沉淀,从而更有效地消化组织。
  7. 孵育细胞悬液15分钟,通过巴氏吸管向上和向下吸移溶液解离细胞悬液。
  8. 重复步骤7一次。
  9. 加入10毫升冰冷的MACS缓冲液,并在4℃下以400×g离心细胞悬浮液4分钟。
  10. 吸出上清液,并在2ml冰冷的渗透裂解缓冲液中重悬细胞沉淀。在室温下孵育4',随后加入10ml冰冷的MACS缓冲液。在4℃下以400×g离心细胞悬浮液7分钟
  11. 重悬细胞在10毫升冰冷的MACS缓冲液,通过一个100微米的细胞过滤器过滤。可以重复使用15ml管收集过滤的细胞。取一个20微升的计数样品,并在4℃,7分钟,4℃下旋转细胞悬浮液的剩余部分。
  12. 加入180微升台盼蓝到20微升的计数样品,混匀。计数单元格。消化后,1个脾通常含有高达60-100×10 6个细胞。
  13. 从细胞沉淀中取出上清液,并以每1ml混合物(见表1)50×10 6个细胞的浓度重悬于抗体混合物I中。在冰上在黑暗中染色20分钟。加入10毫升冰冷的MACS缓冲液,并在4分钟内400转/分4℃下旋转。

    表1. MACS缓冲液中的抗体混合物
    标签
    标记
    克隆
    稀释
    FITC
    CD3
    145-2C11
    1/300
    FITC
    CD19
    1D3
    1/500
    -
    CD16/CD32
    2.4G2
    1/200

  14. 从细胞沉淀中取出上清液,每10×10 6个细胞加入90μl冰冷的MACS缓冲液。
  15. 加入5-10μl/10×10 6个抗-FITC微珠的细胞。 重悬并在冰上孵育15分钟。 加入10ml冰冷的MACS缓冲液,并旋转7分钟400×g 4℃。 弃去上清液。
  16. 根据制造商的协议准备LD柱
  17. 制备后,将15ml管置于LD柱下的冰上
  18. 重悬细胞沉淀在1毫升冰冷的MACS缓冲液和过滤通过100μm柱细胞过滤器在LD柱。
    注意:这最小化细胞凝块阻塞LD柱的概率。 在1ml细胞悬浮液通过后,用2ml冰冷的MACS缓冲液洗涤2次。 确保细胞悬浮液每次通过。 弃去柱子并离心流出液(7分钟400×g/4℃)。 吸出上清液。
  19. 染色细胞沉淀在500μl的抗体混合物II。 在冰上在黑暗中染色20分钟。 加入10ml冰冷的MACS缓冲液,并旋转7分钟400×g 4℃。 吸出上清液并重悬于1ml冰冷的MACS缓冲液中。 将细胞悬液加入到带有细胞过滤帽的5ml聚苯乙烯管中,以除去可能的凝块
    表2. PBS中的抗体mix II
    标签
    标记
    克隆
    稀释
    FITC
    CD3
    145-2C11
    1/300
    FITC
    CD19
    1D3
    1/500
    PE-eFluor610
    CD11c
    N418
    1/300
    PE-Cy5
    CD8a
    57-6.7
    1/600
    PE-Cy7
    CD11b
    M1/70
    1/800
    AF-647
    CD64
    X54-5/7.1
    1/100
    APC-Cy7
    MHCII
    M5/114.15.2
    1/1,000
    eFluor
    506
    生存力
    1/200
    -
    CD16/CD32
    2.4G2
    1/200
    注意:使用PBS对于用可固定生存力染料eFluor 506最佳染色死细胞是必要的。

  20. 通过在2ml PBS中稀释1滴Ultra comp eBeads制备单染色剂。确保在使用前彻底摇动珠子,作为沉淀发生。将200μl的该珠悬浮液加入到7个5ml反应管(6个荧光染料+ 1个未染色的样品)中。与亮荧光团(PE-eFluor610,Pe-Cy5,AF-647)缀合的抗体稀释1/2000,其他抗体稀释1/400。在冰上在黑暗中染色15分钟。加入1ml PBS,并旋转7分钟400×g <4℃。除去上清液,并重悬在200微升PBS的珠子。进入细胞分选器。
    注意:活力染料不与Ultra comp eBeads结合。由于我们排除了死亡细胞(由eFluor 506的阳性染色定义),因此不需要补偿该荧光染料。如果优选补偿,可以用PBS中的eFluor 506活力染料的1/200稀释液对细胞染色。在冰上在黑暗中染色20分钟。用1ml PBS洗涤细胞,并旋转7分钟400×g 4℃。将细胞重悬于200μlPBS中。
  21. 门控设置的示例如下所示。


    图1.脾脏DC的门控策略。FSC-H/FSC-A图中的严格门控策略允许排除双峰。 CD11b + DCs(Lin - > CD11c + MHCII + CD11b + 细胞)包含一部分单核细胞衍生的细胞(具有病原体或炎症的特异性感染可以显着增加这种细胞群体,污染CD11b +细胞)。可以通过排除CD64 + 细胞来鉴定常规的CD11b + DC。

笔记

  1. 人们应该记住尽可能快的工作。一旦从活的动物脾脏DC亚组中移除,则活力快速受损。此外,使用冷试剂改善总生存期
  2. 通过使用剪刀和巴斯德移液管的组织的良好解离和脾细胞在消化培养基中的充分温育对于最佳细胞回收是至关重要的。当调整抗体混合物时,请记住,酶消化可能会切割在感兴趣的细胞类型上表达的表面蛋白。
  3. 在最佳条件下,使用MACS珠的负选择除去90-95%的CD3e +和CD19 +细胞。这将细胞数目减少到大约5-10×10 6个细胞,总排序时间约为20分钟。
  4. 可以从1个脾(在内环境条件下)分选80,000-120,000个CD8a + DC和400,000-600,000个CD11b + CD11b + DC可以通过使用表面标记ESAM或CD4进一步细分。
  5. 在要培养细胞的情况下,科学家应该记住从获得脾脏的时刻开始工作不育(步骤4)。 使用前,可以使用0.2μm过滤器过滤缓冲液以去除潜在的病原体
  6. 可以在加入250ng/ml重组鼠Flt3L的R10培养基中培养细胞。 没有任何进一步的刺激; 这些树突状细胞亚群的存活率在培养24小时后小于60%

食谱

  1. Avertin(40 ml)
    将1克2,2,2-三溴乙醇溶于1毫升2-甲基-2-丁醇中 重悬于39ml PBS中,在室温下振荡过夜 避免直接暴露于光,并在4°C下存储
  2. 2x消化培养基
    用以下命令重建RPMI 1640:
    1/500 DNAse I(10IU/ml) 1/25释放酶TM(0.42Wünsch单位/ml)
  3. MACS缓冲区
    1x PBS
    2%FCS(或2%BSA) 5 mM EDTA
    注意:这通过螯合Ca 2 + 最小化细胞对塑料的粘附。 在0.2μm过滤器上过滤除菌,并在4℃下保存
  4. R10介质
    用以下物质重建RPMI1640-Glutamax:
    10%FBS
    50μg/ml庆大霉素 50μMβ-巯基乙醇
  5. 渗透裂解缓冲液(总共500ml)
    450ml MilliQ水(或其它来源的超纯的ddH 2 O) 4.145g氯化铵
    100μl500mM EDTA
    将pH调节至7.1-7.4
    加入MilliQ水至500 ml
    在0.2μm过滤器上过滤灭菌并在4℃下贮存

致谢

这项工作得到了欧洲研究委员会,欧盟第七框架计划,Fonds Wetenschappelijk Onderzoek Vlaanderen计划(FWO)和Agentschap voor Innovatie door Wetenschap en Technologie(IWT)的支持。

参考文献

  1. Merad,M.,Sathe,P.,Helft,J.,Miller,J.and Mortha,A。(2013)。 树突状细胞谱系:树突状细胞及其子集在稳态和发炎的个体发育和功能 Annu Rev Immunol 31:563-604。
  2. Mebius,R.E.and Kraal,G。(2005)。 脾的结构和功能。 Nat Rev Immunol 5(8):606-616。
  3. Osorio,F.,Tavernier,SJ,Hoffmann,E.,Saeys,Y.,Martens,L.,Vetters,J.,Delrue,I.,De Rycke,R.,Parthoens,E.,Pouliot, Iwawaki,T.,Janssens,S.and Lambrecht,BN(2014)。 展开的蛋白反应传感器IRE-1alpha调节CD8alpha的功能 + < sup> dendritic cells。 Nat Immunol 15(3):248-257。

  • 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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tavernier, S. J., Osorio, F., Janssens, S. and Lambrecht, B. N. (2015). Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting. Bio-protocol 5(5): e1415. DOI: 10.21769/BioProtoc.1415.
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。