搜索

Analysing Temporal Dynamics of T Cell Division in vivo Using Ki67 and BrdU Co-labelling by Flow Cytometry
使用Ki67和BrdU共同标记通过流式细胞术分析体内T细胞分裂的时间动力学   

Thea HoganThea HoganAndrew  YatesAndrew Yates* Benedict  SeddonBenedict Seddon*  (*对本文贡献相同)
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Mar 2017

Abstract

This protocol was developed to increase the richness of information available from in vivo T cell proliferation studies. DNA labelling techniques such as BrdU incorporation allow precise control of label administration and withdrawal, so that the division history of a population can be tracked in detail over long timeframes (days-weeks). Ki67 is expressed in the nucleus of dividing cells, and is retained for a short time (3-4 days) after division (Gossel et al., 2017); therefore acting as a molecular clock to identify cells that have recently divided. Combining these two techniques allows the integration of current and historical proliferation information from individual cells within a population. This data can subsequently be used to probe population dynamics by fitting mathematical models of proliferation (Gossel et al., 2017).

Keywords: T cells (T细胞), Ki67 (Ki67), BrdU labelling (BrdU标记), T cell proliferation (T细胞增殖), DNA labelling (DNA标记), Flow cytometry (流式细胞术)

Background

Quantifying the dynamics of T cell death and division is a significant experimental and computational challenge, yet this information is vital to our understanding of how a healthy immune system develops and is maintained. A variety of techniques exist for in vivo analysis of T cell population dynamics, but the interpretation of data is complicated and results can be model-dependent (Asquith et al., 2002; De Boer and Perelson, 2013). Many of these assays are based on flow cytometric readouts, providing an opportunity to gain novel insights by exploiting the potential for multiparametric analysis. Combining analysis of BrdU uptake alongside Ki67 expression is particularly useful as each represents a distinct parameter of cell division. BrdU is a thymidine analogue that is incorporated into the DNA of dividing cells, and thus permanently marks cells that have divided during the labelling period (Tough and Sprent, 1994; Bonhoeffer et al., 2000). In contrast, Ki67 is a transient marker of cell division: the Ki67 protein is expressed during cell cycle and for a short duration (3-4 days) thereafter (De Boer and Perelson, 2013; Gossel et al., 2017). Measuring Ki67 expression simultaneously with BrdU uptake therefore provides both recent and historical proliferation information from the same cell.

Materials and Reagents

  1. Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150288 )
  2. Nylon mesh (John Staniar & Co., catalog number: NYL9084 )
  3. Pipette tips
  4. Insulin syringes (B. Braun Melsungen, catalog number: 9151125 )
  5. FACS tubes (Corning, Falcon®, catalog number: 352002 )
  6. Mice: adult C57BL/6
  7. FITC BrdU Flow Kit (BD, BD Biosciences, catalog number: 559619 ), containing:
    1. 10 mg/ml BrdU
    2. Cytofix/Cytoperm buffer
    3. 10x Perm/Wash buffer
    4. Cytoperm permeabilisation buffer plus
    5. 1 mg/ml DNase
    6. Anti-BrdU FITC antibody
  8. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190 )
  9. BrdU powder (Sigma-Aldrich, catalog number: B5002 )
  10. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  11. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  12. Anti-mouse Ki67 eFluor660 antibody (clone SolA15) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 50-5698 )
  13. PerCP/Cy5.5 anti-mouse TCR beta chain (clone H57-597) (BioLegend, catalog number: 109228 )
  14. Brilliant Violet 711 anti-mouse CD4 (clone RM4-5) (BioLegend, catalog number: 100550 )
  15. Brilliant Violet 510 anti-mouse CD25 (clone PC61) (BioLegend, catalog number: 102042 )
  16. Brilliant Violet 785 anti-mouse/human CD44 (clone IM7) (BioLegend, catalog number: 103059 )
  17. BD Horizon BUV737 rat anti-mouse CD62L (clone MEL-14) (BD, BD Biosciences, catalog number: 565213 )
  18. LIVE/DEAD fixable near-IR dead cell stain kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: L10119 )
  19. 25x BrdU/Sucrose (see Recipes)
  20. PBS/BSA (see Recipes)
  21. Surface Marker Antibody Cocktail (see Recipes)
  22. 1x Perm/Wash buffer (see Recipes)
  23. DNase (see Recipes)

Equipment

  1. Pipettes
  2. Dissection tools (sharp scissors and forceps)
  3. Incubator
  4. Refrigerated bench top centrifuge
  5. Cell counter, for example a CASY counter (Omni Life Science, catalog number: 5651697 )
  6. Flow cytometer equipped with appropriate lasers and filters for detection of FITC and eFluor660 fluorescence

Procedure

  1. In vivo administration of BrdU
    Notes:
    1. BrdU is initially administered by intraperitoneal injection to enable precise timing of the start of administration. Following this initial injection, mice are maintained on BrdU administered via the drinking water, to avoid undue stress that might be caused by repeated injections.
    2. A mouse that has not been fed BrdU should also be included, to allow isolation of BrdU-unlabelled cells from the same population to be used as a negative control to determine background levels of staining for the anti-BrdU antibody. 

    1. Prepare a working solution of 4 mg/ml BrdU by diluting 10 mg/ml BrdU stock solution (provided in BrdU FITC Flow Kit) in sterile PBS.
      Note: This can be prepared in advance and aliquots stored at -20 °C for up to two months. Protect from light and avoid repeated freeze-thaw cycles.
    2. Inject 200 μl of BrdU working solution (i.e., 0.8 mg BrdU) per mouse via the intraperitoneal route.
    3. Dilute 25x BrdU/Sucrose (see Recipes) to 1x in mouse drinking water. The final concentration in drinking water is 0.8 mg/ml BrdU and 0.5% sucrose.
      Note: Water bottles should be protected from light. Sucrose is included here to ensure palatability of the drinking water containing BrdU. BrdU has a limited stability in drinking water and must therefore be refreshed regularly to maintain the dosage level.
    4. Maintain BrdU in drinking water for the desired timeframe, refreshing every 2-3 days.
    5. To withdraw BrdU treatment for a delabelling timecourse, at the required timepoint replace the water bottle containing BrdU with a bottle containing normal drinking water.

  2. Co-staining with BrdU and Ki67 for flow cytometry analysis
    Note: Refer to the manufacturer’s instructions in the FITC BrdU Flow Kit. Keep samples on ice (except where indicated) and protect from light.
    1. Day 1. Staining for cell surface markers, fixation and permeabilisation
      1. Euthanise BrdU-treated mice by cervical dislocation and remove the organs containing the populations of interest for analysis–for example, lymph nodes.
      2. Place the lymph nodes onto a piece of nylon mesh in a Petri dish containing 3 ml of PBS/BSA (see Recipes). Cover with a second piece of nylon mesh and use forceps to apply pressure to this top piece of mesh, gently crushing the organs and releasing the cells into the buffer. Use a pipette to collect the 3 ml of buffer containing the cells in suspension and transfer this cell suspension to a FACS tube.
      3. Perform cell counts and transfer 2 million total cells to FACS tubes for staining.
        Notes:
        1. CD4 effector memory T cells are approximately 1% of total lymph node cells. Staining 2 million total lymph node cells typically permits analysis of at least 10,000 CD4 effector memory T cells.
        2. It may be possible to stain fewer cells using this protocol, using the same volumes indicated for 2 million cells, but this has not been tested.
      4. Wash cells by adding 1 ml of PBS/BSA to each tube and centrifuge at 300 x g for 5 min at 4 °C. Discard supernatant.
      5. Resuspend cells in 100 μl of Surface Marker Antibody Cocktail (see Recipes) and incubate for 1 h on ice.
        Notes:
        1. The surface marker staining panel described here was used for the identification of CD4 effector memory cells in mouse lymph nodes, but could be adapted as required to study other populations of interest.
        2. Staining controls using cells from BrdU treated mice should be processed alongside experimental samples. Staining controls should include: single colour controls for each antibody used, an unstained control, and a fluorescence-minus-one control for Ki67-eFluor660. Additionally, cells from a mouse not fed BrdU should be stained with all antibodies as a negative control to determine background levels of staining for the anti-BrdU antibody.
      6. Wash cells by adding 1 ml of PBS/BSA to each tube and centrifuge at 300 x g for 5 min at 4 °C. Discard supernatant.
      7. Fix and permeabilise cells by resuspending in 100 μl Cytofix/Cytoperm buffer (provided in FITC BrdU Flow Kit) and incubate for 30 min on ice.
      8. Wash cells by adding 1 ml freshly prepared 1x Perm/Wash buffer (see Recipes), centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      9. Resuspend cells in 1 ml PBS/BSA and store at 4 °C overnight.
        Note: Overnight storage is not mandatory, but may be preferable when processing large numbers of samples. Proceed directly from step B1f to step B2c if overnight storage is not required.
    2. Day 2. Co-staining for BrdU and Ki67
      1. Recover cells from storage and centrifuge at 300 x g for 5 min at 4 °C, discard supernatant.
      2. Wash cells by resuspending pellet in 1 ml of freshly prepared 1x Perm/wash buffer, centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      3. Resuspend cells in 100 μl Cytoperm Permeabilisation Buffer Plus (provided in FITC BrdU Flow Kit) and incubate for 10 min on ice.
      4. Wash the Cytoperm Permeabilisation Buffer Plus off the cells by adding 1 ml of freshly prepared 1x Perm/Wash buffer, centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      5. Repeat fixation by resuspending cells in 100 μl of Cytofix/Cytoperm buffer and incubate for 5 min at room temperature.
      6. Wash cells by adding 1 ml of freshly prepared 1x Perm/Wash buffer, centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      7. Expose incorporated BrdU by resuspending cells in 100 μl of 300 μg/ml DNase (see Recipes) and incubate for 1 h at 37 °C.
      8. Wash cells by adding 1 ml of freshly prepared 1x Perm/Wash buffer, centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      9. Co-stain for BrdU and Ki67 by resuspending cells in 50 μl 1x Perm/Wash containing anti-BrdU FITC (provided in FITC BrdU Flow Kit) at a dilution of 1:50 and anti-Ki67 eFluor660 at a dilution of 1:200. Incubate for 1 h at room temperature.
      10. Wash cells by adding 1 ml of freshly prepared 1x Perm/Wash buffer, centrifuge at 300 x g for 5 min at 4 °C and discard supernatant.
      11. Repeat wash to ensure all residual unbound antibody is removed from cells.
      12. Acquire samples on a flow cytometer equipped with appropriate lasers and filters for the detection of FITC and eFluor660 fluorescence.

Data analysis

  1. Using appropriate flow cytometry analysis software, gate on population of interest (e.g., TCR+CD4+CD25-CD44hiCD62Llo for CD4 effector memory T cells in Figure 1A) and display a two dimensional dotplot of Ki67 vs. BrdU (Figure 1B).
  2. Apply quadrant gates as shown in Figure 1B, using a BrdU untreated mouse as a negative control for BrdU staining and a fluorescence-minus-one control for Ki67 eFluor660 staining. Cells will fall into a given quadrant depending on their division history, as described in Figure 1B.


    Figure 1. Example flow cytometry analysis (from Gossel et al., 2017). Representative flow cytometry plots showing (A) gating strategy for CD4 effector memory T cells (TCR+CD4+CD25-CD44hiCD62Llo) in lymph nodes, and (B) Ki67 and BrdU in CD4 effector memory T cells from the lymph nodes of mice treated with BrdU for either one day (left) or 21 days (right). Quadrant gates represent the division history of cells as follows: Ki67+BrdU- cells have recently divided (Ki67 lifetime 3-4 days) but have not incorporated BrdU. This may be due to either inefficient uptake of BrdU, or because the cell division did not take place within the BrdU treatment window. Ki67+BrdU+ cells have recently divided (Ki67 lifetime 3-4 days), and this cell division has taken place within the BrdU treatment window. Ki67-BrdU+ cells have divided at some time during the BrdU treatment window, but the lack of Ki67 expression indicates that this cell division was not recent (Ki67 lifetime 3-4 days). Ki67-BrdU- cells have either not divided at all, or have divided but subsequently lost expression of Ki67 (lifetime 3-4 days) and/or BrdU (due to either inefficient uptake of BrdU or label dilution through repeated cell divisions). Numbers indicate the percentage of cells within each quadrant.

  3. Determine the fraction of cells that have incorporated BrdU within the Ki67+ and Ki67- subpopulations as described in Figure 2, which shows example data for BrdU labelling and delabelling timecourses in the CD4 effector memory population.
  4. The data generated in step 3 can subsequently be used to fit mathematical models of T cell proliferation as described in Gossel et al. (2017).


    Figure 2. Example timecourse data (from Gossel et al., 2017). Graphs show the fraction of BrdU+ cells in either the Ki67+ or Ki67- subpopulation of CD4 effector memory T cells (TCR+CD4+CD25-CD44hiCD62Llo) in mouse lymph nodes. Each point represents one mouse. Mice were fed BrdU continuously for the indicated time periods up to 21 days. After 21 days, BrdU treatment was withdrawn and the delabelling timecourse was followed for a further 14 days. The fraction of BrdU+ cells within the Ki67+ subpopulation (top) is given by the formula B/(A+B) where A and B refer to the Ki67+BrdU- and Ki67+BrdU+ quadrants. The fraction of BrdU+ cells within the Ki67-+ subpopulation (bottom) is given by the formula C/(C+D) where C and D refer to the Ki67-BrdU+ and Ki67-BrdU- quadrants. These data can subsequently be used to fit mathematical models of T cell proliferation as described in Gossel et al. (2017).

Notes

  1. BrdU is administered to mice via an initial intraperitoneal injection of 0.8 mg, to ensure precise control of the start time. Thereafter, BrdU is maintained in drinking water at 0.8 mg/ml for the desired time up to 21 days. Sucrose is also added at 0.5% to the drinking water to ensure palatability. For delabelling timecourses, mice are switched back to regular drinking water at the required timepoint.
  2. To eliminate variability caused by processing samples on different days, it is preferable to stagger the start times for different groups so that all mice in an experiment finish their time course on the same day. As an example, for a timecourse of one, two and three days on BrdU, one group of mice would start BrdU treatment on Monday, the next group on Tuesday, and the third group on Wednesday, with all mice in all groups sacrificed together on Thursday.
  3. It should be noted that this protocol is optimized for the concurrent staining of BrdU and Ki67 within the same cell. The fixation protocol and concentration of antibody used for Ki67 eFluor660 differ significantly from the manufacturer’s recommendations for staining with this antibody alone. Any clone and/or fluorochrome substitutions should be thoroughly and carefully tested before use.

Recipes

  1. 25x BrdU/Sucrose
    1. Add BrdU powder to a concentration of 20 mg/ml in distilled water which has been warmed to 37 °C
    2. Stir gently, protected from light, until dissolved
    3. Add sucrose to 12.5%
    Note: This can be prepared in advance and single-use aliquots stored at -20 °C for up to two months, protected from light.
  2. PBS/BSA
    1. Supplement PBS with 1% BSA
    2. Filter sterilise and store at 4 °C for up to 1 month
  3. Surface Marker Antibody Cocktail–dilute antibodies to required concentration in PBS
    1. PerCP/Cy5.5 anti TCR–dilute to 0.5 μg/ml
    2. BV711 anti CD4–dilute to 0.25 μg/ml
    3. BV510 anti CD25–dilute to 0.25 μg/ml
    4. BV785 anti CD44–dilute to 0.5 μg/ml
    5. BUV737 anti CD62L–dilute to 0.25 μg/ml
    6. Add 1 μl of LIVE/DEAD near-IR dye per 800 μl of surface marker antibody cocktail
  4. 1x Perm/Wash buffer
    Dilute 10x Perm/Wash buffer (provided in FITC BrdU Flow Kit) 1:10 in distilled water
    Prepare fresh each time
  5. DNase
    Dilute 1 mg/ml DNase (provided in FITC BrdU Flow Kit) in PBS to a final concentration of 300 μg/ml

Acknowledgments

This protocol was developed for a published study (Gossel et al., 2017), supported by the National Institutes of Health (R01 AI093870) and the Medical Research Council (MC-PC-13055). The authors declare no conflicts of interest or competing interests.

References

  1. Asquith, B., Debacq, C., Macallan, D. C., Willems, L. and Bangham, C. R. (2002). Lymphocyte kinetics: the interpretation of labelling data. Trends Immunol 23(12): 596-601.
  2. Bonhoeffer, S., Mohri, H., Ho, D. and Perelson, A. S. (2000). Quantification of cell turnover kinetics using 5-bromo-2'-deoxyuridine. J Immunol 164(10): 5049-5054.
  3. De Boer, R. J. and Perelson, A. S. (2013). Quantifying T lymphocyte turnover. J Theor Biol 327: 45-87.
  4. Gossel, G., Hogan, T., Cownden, D., Seddon, B. and Yates, A. J. (2017). Memory CD4 T cell subsets are kinetically heterogeneous and replenished from naive T cells at high levels. Elife 6.
  5. Tough, D. F. and Sprent, J. (1994). Turnover of naive- and memory-phenotype T cells. J Exp Med 179(4): 1127-1135.

简介

该协议的开发是为了增加从体内T细胞增殖研究中获得的信息的丰富性。 诸如BrdU掺入的DNA标记技术允许精确控制标记施用和撤回,从而可以在很长时间段(日 - 周)内详细地追踪群体的分裂历史。 Ki67在分裂细胞的细胞核中表达,并在分裂后短时间(3-4天)保留(Gossel等人,2017)。 因此充当分子时钟来识别最近分裂的细胞。 将这两种技术结合起来就可以整合来自个体细胞的当前和历史扩散信息。 这个数据随后可以用来通过拟合增殖的数学模型来探测种群动态(Gossel et al。,2017)。


【背景】量化T细胞死亡和分裂的动力学是一个重大的实验和计算挑战,但是这个信息对于我们理解一个健康的免疫系统如何发展和维持至关重要。 T细胞群体动态的体内分析有多种技术,但数据的解释是复杂的,结果可能是模型依赖的(Asquith et al。2002 ; De Boer和Perelson,2013)。许多这些分析基于流式细胞仪读数,通过利用多参数分析的潜力提供获得新颖见解的机会。 BrdU吸收与Ki67表达一起的分析结合是特别有用的,因为每个代表细胞分裂的独特参数。 BrdU是掺入分裂细胞DNA中的胸苷类似物,因此永久标记在标记期间已分裂的细胞(Tough和Sprent,1994; Bonhoeffer等人,2000)。相比之下,Ki67是细胞分裂的暂时性标记:Ki67蛋白在细胞周期中表达并且此后短时间(3-4天)表达(De Boer和Perelson,2013; Gossel等人,2017)。测量Ki67表达与BrdU摄取同时提供来自同一细胞的最近和历史的增殖信息。

关键字:T细胞, Ki67, BrdU标记, T细胞增殖, DNA标记, 流式细胞术

材料和试剂

  1. 培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150288)
  2. 尼龙网(John Staniar& Co.,目录号:NYL9084)
  3. 移液器提示
  4. 胰岛素注射器(B.BraunMelsungen,目录号:9151125)
  5. FACS管(Corning,Falcon ,产品目录号:352002)
  6. 小鼠:成人C57BL / 6
  7. FITC BrdU Flow Kit(BD,BD Biosciences,目录号:559619),包含:
    1. 10毫克/毫升BrdU
    2. Cytofix / Cytoperm缓冲液
    3. 10倍Perm /清洗缓冲液
    4. 细胞渗透缓冲液加
    5. 1毫克/毫升DNase
    6. 抗BrdU FITC抗体
  8. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190)
  9. BrdU粉末(Sigma-Aldrich,目录号:B5002)
  10. 蔗糖(Sigma-Aldrich,目录号:S0389)
  11. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  12. 抗小鼠Ki67 eFluor660抗体(克隆SolA15)(Thermo Fisher Scientific,eBiosciences TM,目录号:50-5698)
  13. PerCP / Cy5.5抗小鼠TCRβ链(克隆H57-597)(BioLegend,目录号:109228)
  14. Brilliant Violet 711抗小鼠CD4(克隆RM4-5)(BioLegend,目录号:100550)
  15. Brilliant Violet 510抗小鼠CD25(克隆PC61)(BioLegend,目录号:102042)
  16. Brilliant Violet 785抗小鼠/人CD44(克隆IM7)(BioLegend,目录号:103059)
  17. BD Horizon BUV737大鼠抗小鼠CD62L(克隆MEL-14)(BD,BD Biosciences,目录号:565213)
  18. LIVE / DEAD可固定近红外死细胞染色试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:L10119)
  19. 25倍BrdU /蔗糖(见食谱)
  20. PBS / BSA(见食谱)
  21. 表面标记抗体鸡尾酒(见食谱)
  22. 1倍Perm /洗涤缓冲液(见食谱)
  23. DNase(见食谱)

设备

  1. 移液器
  2. 解剖工具(锋利的剪刀和镊子)
  3. 孵化器
  4. 冷冻台式离心机
  5. 细胞计数器,例如CASY计数器(Omni Life Science,目录号:5651697)
  6. 流式细胞仪配备适当的激光和过滤器检测FITC和eFluor660荧光

程序

  1. BrdU体内管理
    注意:
    1. BrdU最初通过腹膜内注射施用以确保开始施用的精确时间。在初次注射后,通过饮用水将小鼠维持在BrdU上,以避免可能由反复注射引起的不必要的压力。
    2. 还应该包括尚未饲喂BrdU的小鼠,以允许从相同群体中分离BrdU-未标记的细胞作为阴性对照以确定抗BrdU抗体的染色背景水平。 EM> 

    1. 通过在无菌PBS中稀释10mg / ml BrdU储备溶液(在BrdU FITC Flow Kit中提供)来制备4mg / ml BrdU的工作溶液。
      注意:可以事先准备好,等分试样在-20°C保存两个月。避免光照,避免反复的冻融循环。
    2. 每只小鼠腹腔注射200μlBrdU工作液(即,0.8mg BrdU)。
    3. 在小鼠饮用水中稀释25x BrdU /蔗糖(见食谱)至1x。饮用水中的最终浓度是0.8mg / ml BrdU和0.5%蔗糖。
      注意:水瓶应避光。此处包括蔗糖以确保含有BrdU的饮用水的适口性。 BrdU在饮用水中的稳定性有限,因此必须定期更新以维持剂量水平。
    4. 在所需的时间内保持饮用水中的BrdU,每2-3天更新一次。
    5. 为了取消BrdU处理标记的时间进程,在规定的时间点,用装有普通饮用水的瓶子取代装有BrdU的水瓶。

  2. 用BrdU和Ki67共染色进行流式细胞仪分析
    注意:请参阅FITC BrdU Flow Kit中的制造商说明。将样品保存在冰上(除非另有说明)并避光保存。
    1. 第1天:染色细胞表面标记,固定和透化
      1. 安乐死BrdU治疗的小鼠颈椎脱臼,并移除含有感兴趣的人群的器官进行分析,例如淋巴结。
      2. 将淋巴结放在含有3ml PBS / BSA的培养皿中的尼龙网上(参见食谱)。用第二块尼龙网覆盖,用镊子将压力施加到该顶部网片上,轻轻压碎器官并将细胞释放到缓冲液中。用移液管收集含有悬浮细胞的3ml缓冲液,并将该细胞悬液转移到FACS管中。
      3. 进行细胞计数,并将200万个总细胞转移至FACS管进行染色。
        注意:
        1. CD4效应记忆T细胞约占总淋巴结细胞的1%。染色200万总淋巴结细胞通常允许至少10,000个CD4效应记忆T细胞的分析
        2. 使用此方案染色较少的细胞可能是可能的,使用与2百万个细胞相同的体积,但尚未经过测试。
      4. 通过向每个管中加入1ml PBS / BSA来洗涤细胞,并在4℃下以300gxg离心5分钟。丢弃上清。
      5. 重悬细胞100μL表面标记抗体鸡尾酒(见食谱),并在冰上孵育1小时。
        注意:
        1. 这里描述的表面标记染色小组用于鉴定小鼠淋巴结中的CD4效应记忆细胞,但是可以根据需要进行调整以研究其他感兴趣的群体。
        2. 使用来自BrdU处理的小鼠的细胞的染色对照应该与实验样品一起处理。染色对照应包括:使用的每种抗体的单一颜色对照,Ki67-eFluor660的未染色对照和荧光减一对照。此外,来自未饲喂BrdU的小鼠的细胞应当用作为阴性对照的所有抗体进行染色,以确定抗BrdU抗体染色的背景水平。
      6. 通过向每个管中加入1ml PBS / BSA来洗涤细胞,并在4℃下以300gxg离心5分钟。丢弃上清。
      7. 通过重新悬浮于100μlCytofix / Cytoperm缓冲液(在FITC BrdU Flow Kit中提供)并在冰上孵育30分钟来固定和透化细胞。
      8. 通过加入1ml新鲜制备的1x Perm /洗涤缓冲液(参见食谱)来洗涤细胞,在4℃下以300gxg离心5分钟并丢弃上清液。
      9. 在1毫升的PBS / BSA重悬细胞,并储存在4°C过夜。
        注意:隔夜存储不是强制性的,但在处理大量样本时可能更好。如果不需要隔夜存储,则直接从步骤B1f继续步骤B2c。
    2. 第2天。BrdU和Ki67的共染色
      1. 从储存中回收细胞,并在4℃下300×g离心5分钟,弃去上清液。
      2. 通过将沉淀重新悬浮于1ml新鲜制备的1x Perm /洗涤缓冲液中来洗涤细胞,在4℃下以300gxg离心5分钟并弃去上清液。
      3. 在100μlCytoperm Permeabilisation Buffer Plus(在FITC BrdU Flow Kit中提供)中重悬细胞并在冰上孵育10分钟。
      4. 通过加入1ml新鲜制备的1x Perm /洗涤缓冲液洗涤细胞渗透缓冲液加上离开细胞,在4℃下300xg离心5分钟并丢弃上清液。
      5. 通过将细胞重悬于100μlCytofix / Cytoperm缓冲液中并在室温下孵育5分钟来重复固定。
      6. 通过加入1ml新制备的1x Perm / Wash缓冲液来洗涤细胞,在4℃下以300gxg离心5分钟并丢弃上清液。
      7. 通过将细胞重悬于100μl的300μg/ ml DNA酶(参见食谱)中并且在37℃孵育1小时来暴露掺入的BrdU。
      8. 通过加入1ml新制备的1x Perm / Wash缓冲液来洗涤细胞,在4℃下以300gxg离心5分钟并丢弃上清液。
      9. BrdU和Ki67的共同染色是通过将细胞重悬浮在含有以1:50稀释的抗BrdU FITC(在FITC BrdU Flow Kit中提供)的50μl1x Perm / Wash和稀释度为1:200的抗Ki67 eFluor660中。
        在室温下孵育1小时
      10. 通过加入1ml新制备的1x Perm / Wash缓冲液来洗涤细胞,在4℃下以300gxg离心5分钟并丢弃上清液。
      11. 重复洗涤,以确保所有残留的未结合的抗体从细胞中移除。
      12. 在配备适当的激光和过滤器的流式细胞仪上采集样品,以检测FITC和eFluor660荧光。

数据分析

  1. 使用合适的流式细胞术分析软件,在感兴趣的群体(例如,TCR + CD4 + CD25 - CD44图1A中的CD4效应记忆T细胞的CD62L )和显示Ki67与BrdU的二维点图(图1B)。
  2. 如图1B所示应用象限门,使用BrdU未处理的小鼠作为BrdU染色的阴性对照,Ki67 eFluor660染色的荧光减1对照。
    如图1B所示,细胞将落入一个给定的象限,取决于它们的分裂历史

    图1.流式细胞仪分析实例(来自Gossel等人,2017)效应记忆T细胞(TCR + CD4 + CD25 + CD44 + CD62L lo) ),以及(B)来自用BrdU处理的小鼠的淋巴结的CD4效应记忆T细胞的一天(左)或21天(右)的Ki67和BrdU。象限门代表细胞的分裂历史如下:Ki67 + BrdU-细胞最近分裂(Ki67寿命3-4天),但没有掺入BrdU。这可能是由于BrdU吸收不足,或者由于细胞分裂不在BrdU治疗窗口内发生。最近将Ki67 + BrdU +细胞分裂(Ki67寿命3-4天),并且该细胞分裂在BrdU处理窗口内发生。 Ki67-BrdU +细胞在BrdU治疗期间的某个时间段已经分裂,但Ki67表达的缺乏表明这种细胞分裂不是最近的(Ki67寿命3-4天)。 Ki67-BrdU-细胞根本没有分裂,或者已经分裂但随后失去了表达Ki67(寿命3-4天)和/或BrdU(由于BrdU的低效吸收或通过重复的细胞分裂而标记稀释)。数字表示每个象限内的细胞百分比。

  3. 如图2所示,确定在Ki67 +和Ki67-亚群内掺入BrdU的细胞比例,其显示了CD4效应物记忆群体中BrdU标记和去标记时间进程的实例数据。
  4. 随后可将步骤3中产生的数据用于拟合T细胞增殖的数学模型,如Gossel等人(2017)。


    图2.示例性时间历程数据(来自Gossel等人,2017)。图显示Ki67 +或Ki67中BrdU +细胞的分数 - CD4效应记忆T细胞亚群(TCR + CD4 + CD25-CD44 hi CD62L lo) )在小鼠淋巴结。每个点代表一只老鼠。 BrdU小鼠连续饲喂指定的时间至21天。在21天后,BrdU治疗被撤销,并且标记时间进程被追踪了14天。 Ki67 +亚群(顶部)中的BrdU +细胞部分由公式B /(A + B)给出,其中A和B指Ki67 + BrdU-和Ki67 + BrdU +象限。 Ki67- +亚群(底部)中的BrdU +细胞部分由公式C /(C + D)给出,其中C和D指Ki67-BrdU +和Ki67-BrdU-象限。这些数据可以随后用于拟合Gossel等人(2017)中描述的T细胞增殖的数学模型。

笔记

  1. BrdU通过初始腹腔注射0.8mg施用给小鼠以确保精确控制开始时间。此后,将BrdU保持在0.8mg / ml的饮用水中达所需时间至21天。蔗糖也以0.5%添加到饮用水中以确保适口性。为了标记时间进程,小鼠在规定的时间点被转换回常规饮用水。
  2. 为了消除在不同日子处理样品引起的变异性,优选错开不同组的开始时间,使得实验中的所有小鼠在同一天完成其时间过程。例如,在BrdU的一天,两天和三天的时间里,一组老鼠将在星期一开始BrdU治疗,在星期二开始下一组,在星期三开始第三组,所有组的所有老鼠一起牺牲星期四。
  3. 应该注意的是,该方案针对同一细胞内BrdU和Ki67的同时染色进行了优化。用于Ki67 eFluor660的抗体的固定方案和浓度与制造商关于单独使用该抗体染色的建议显着不同。
    任何克隆和/或荧光物质替代应在使用前彻底和仔细测试

食谱

  1. 25x BrdU /蔗糖

    1. 在加热到37℃的蒸馏水中加入浓度为20 mg / ml的BrdU粉末
    2. 轻轻搅拌,避光,直至溶解
    3. 添加蔗糖至12.5%
    注意:这可以预先准备好,一次性使用的等分试样在-20°C保存两个月,避光。
  2. PBS / BSA
    1. 补充PBS 1%BSA
    2. 过滤消毒,并在4°C储存长达1个月
  3. 表面标记抗体鸡尾酒稀释抗体在PBS中的所需浓度
    1. PerCP / Cy5.5抗TCR-稀释到0.5μg/ ml
    2. BV711抗CD4-稀释至0.25μg/ ml
    3. BV510抗CD25-稀释至0.25μg/ ml
    4. BV785抗CD44-稀释到0.5μg/ ml
    5. BUV737抗CD62L-稀释至0.25μg/ ml

    6. 每800μl添加1μlLIVE / DEAD近红外染料
  4. 1倍Perm /清洗缓冲液
    用蒸馏水稀释10倍Perm / Wash缓冲液(FITC BrdU Flow Kit中提供)1:10
    准备每次新鲜
  5. DNase
    在PBS中稀释1mg / ml DNA酶(在FITC BrdU Flow Kit中提供)至终浓度为300μg/ ml

致谢

该协议是为由国立卫生研究院(R01 AI093870)和医学研究委员会(MC-PC-13055)支持的已发表的研究(Gossel等人,2017)开发的。作者声明不存在利益冲突或利益冲突。

参考

  1. Asquith,B.,Debacq,C.,Macallan,D.C.,Willems,L。和Bangham,C.R。(2002)。 淋巴细胞动力学:标记数据的解释。趋势免疫 23(12):596-601。
  2. Bonhoeffer,S.,Mohri,H.,Ho,D。和Perelson,A.S。(2000)。 使用5-溴-2'-脱氧尿苷定量细胞更新动力学 J Immunol 164(10):5049-5054。
  3. De Boer,R. J.和Perelson,A. S.(2013)。 量化T淋巴细胞转换。 J Theor Biol 327: 45-87。
  4. Gossel,G.,Hogan,T.,Cownden,D.,Seddon,B。和Yates,A.J。(2017)。记忆性CD4 T细胞亚型动力学异质性,并从高水平的天真T细胞补充。 > Elife 6.
  5. 坚韧,D.F。和Sprent,J。(1994)。 幼稚和记忆型表型T细胞的转换 J Exp Med 179(4):1127-1135。
  • 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 Hogan et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hogan, T., Yates, A. and Seddon, B. (2017). Analysing Temporal Dynamics of T Cell Division in vivo Using Ki67 and BrdU Co-labelling by Flow Cytometry. Bio-protocol 7(24): e2649. DOI: 10.21769/BioProtoc.2649.
  2. Gossel, G., Hogan, T., Cownden, D., Seddon, B. and Yates, A. J. (2017). Memory CD4 T cell subsets are kinetically heterogeneous and replenished from naive T cells at high levels. Elife 6.
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的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。