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Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics
生成白消安嵌合体小鼠用于T细胞群体动力学分析   

Thea  HoganThea HoganAndrew  YatesAndrew Yates* Benedict  SeddonBenedict Seddon*  (*对本文贡献相同)
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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 generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), with T-cell depleted donor bone marrow bearing a different congenic marker (CD45.2) to that of the host mouse (CD45.1). New cell production post-BMT can thus be tracked by measuring the fraction of CD45.2+ cells over time within a population of interest (Hogan et al., 2015; Gossel et al., 2017).

Keywords: T cells (T细胞), T cell homeostasis (T细胞稳态), Bone marrow chimeras (骨髓嵌合体), Busulfan (白消安), Temporal fate mapping (时间命运映射)

Background

Bone marrow chimeras are a valuable tool for studying immune system development and function. Typically, chimeras are generated by irradiation of host mice followed by transplantation with donor bone marrow. Irradiation causes considerable damage to the haematopoietic system, and full immune reconstitution is delayed by weeks to months post-transplant (Fry and Mackall, 2005). The resulting period of lymphopenia drives spontaneous proliferation of naïve T cells and the acquisition of a memory-like phenotype (Goldrath et al., 2004). To avoid the perturbation of immune homeostasis caused by irradiation, we turned our attention to the conditioning drug busulfan. Busulfan is an alkylating agent that is toxic to haematopoietic stem cells (HSC) but does not deplete circulating lymphocytes (Westerhof et al., 2000; Hsieh et al., 2007). Following busulfan conditioning and bone marrow transplantation (BMT), chimeras are indistinguishable from untreated controls: they have normal numbers of naïve and memory CD4 and CD8 T cells, and these cells express normal levels of the proliferation marker Ki67 (Hogan et al., 2015; Gossel et al., 2017). Therefore, busulfan conditioning allows induction of chimerism while preserving peripheral immune homeostasis. By using donor bone marrow bearing a congenic marker, it is possible to distinguish host- or donor-origin cells by flow cytometry. While HSC replacement is never absolute, emergence of donor origin cells can be used as an accurate proxy for de novo haematopoetic development since BMT, thereby allowing cells within a population to be stratified by age on the basis of congenic marker expression.

Materials and Reagents

  1. 5 ml syringes (BD, catalog number: 302187 )
  2. 25 G needles (Terumo, catalog number: GS-351 )
  3. Insulin syringes (B. Braun Melsungen, catalog number: 9151125 )
  4. Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150288 )
  5. 70 μm cell strainers (Corning, Falcon®, catalog number: 352350 )
  6. 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
  7. FACS tubes with lid (Corning, Falcon®, catalog number: 352058 )
  8. Host mice: B6 CD45.1 adult females (THE JACKSON LABORATORY, catalog number: 002014 )
  9. Donor mice: C57BL/6 CD45.2 adult females (THE JACKSON LABORATORY, catalog number: 000664 )
    Note: One donor typically provides sufficient T-cell depleted bone marrow for two recipients.
  10. Busilvex® 6 mg/ml busulfan concentration for infusion (Pierre Fabre)
  11. Biotinylated anti-TCRβ (Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-5961 )
  12. Biotinylated anti-CD3ε (Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-0031 )
  13. Dynabeads M-280 streptavidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11206D )
  14. Fluorescent streptavidin (for example, Streptavidin APC, BioLegend, catalog number: 405207 )
  15. Fluorescent antibody to CD45.1 (for example, anti-CD45.1 Brilliant Violet 650, BioLegend, catalog number: 110736 )
  16. Fluorescent antibody to CD45.2 (for example, anti-CD45.2 PE-Dazzle 594, BioLegend, catalog number: 109846 )
  17. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190 )
  18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  19. PBS/BSA (see Recipes)

Equipment

  1. Dissection tools (sharp scissors and forceps)
  2. Refrigerated benchtop centrifuge
  3. EasySepTM magnet (STEMCELL Technologies, catalog number: 18000 )
  4. Tube rotator
  5. Flow cytometer

Procedure

Note: The steps in this procedure are summarised in Figure 1. All animal procedures were performed in accordance with UK Home Office regulations.


Figure 1. Generation of busulfan chimeric mice. This schematic summarises the principal steps of this protocol for the generation of busulfan chimeric mice. Bone marrow cells from CD45.2+ donor mice are labeled with biotinylated antibodies to CD3 and TCRb, followed by labelling with streptavidin-coated magnetic beads. Labelled cells are immobilised using a magnet, allowing the unbound fraction to be collected. T cell depleted bone marrow cells are then injected into busulfan-conditioned CD45.1+ host mice.

  1. Busulfan treatment of host CD45.1 mice (Day -2 and Day -1)
    1. Prepare a fresh working solution of 1 mg/ml busulfan by diluting Busilvex (6 mg/ml busulfan) in sterile PBS.
      Note: This must be prepared fresh each day and cannot be stored.
    2. Ear mark and weigh each mouse individually prior to each injection and calculate the appropriate volume of working solution required to deliver a dose of 10 μg/g busulfan–i.e., 10 μl of 1 mg/ml working solution per gram of body weight.
    3. Day -2: Deliver appropriate dose of busulfan by intraperitoneal injection.
    4. Allow host mice 24 h recovery after injection.
    5. Day -1: Repeat Steps A1-A4 for a second injection of busulfan. The final dose of busulfan in each mouse is thus 20 μg/g, delivered as two doses of 10 μg/g 24 h apart.
      Note: Mice remain healthy and alert after busulfan injection. HSC are depleted within 24 h after busulfan treatment (Westerhof et al., 2000).

  2. Preparation of T cell depleted donor CD45.2 bone marrow (Day 0)
    Note: Cell suspensions can be prepared on the bench; a laminar flow hood is not required.
    1. Euthanise donor mice by cervical dislocation and remove the femur and tibia from both hind legs.
    2. Use a 5 ml syringe fitted with a 25 G needle to flush each of the bones with PBS/BSA (see Recipes), collecting the marrow in a Petri dish.
    3. Filter the bone marrow through a 70 μm cell strainer into a 50 ml tube.
    4. Centrifuge at 300 x g for 5 min at 4 °C, discard supernatant and resuspend in 1 ml PBS/BSA per donor mouse.
    5. Count cells (expect to recover ~40 million cells per donor mouse) and adjust the volume of cell suspension to a concentration of 20-40 million cells/ml.
    6. Add anti-TCRβ and anti-CD3ε biotinylated antibodies to a final concentration of 1 μg/ml for each, incubate with rotation for 30 min at 4 °C.
    7. Centrifuge at 300 x g for 5 min at 4 °C, discard supernatant and resuspend in PBS/BSA at a concentration of at 20-40 million cells/ml.
    8. Transfer cell suspension to 5 ml FACS tubes with lids. Use multiple tubes if necessary, with a volume of 3-4 ml per tube.
    9. Add streptavidin Dynabeads to a bead: cell ratio of approximately 1:1 and incubate with rotation for 30 min at 4 °C.
    10. Insert the tube of beads with cell suspension into the Easysep magnet and incubate for 2 min on ice.
    11. Collect the supernatant containing the unbound fraction of cells into a fresh tube.
    12. Repeat Steps B10-B11 with the collected unbound fraction of cells, to ensure all beads are removed from the cell suspension.
    13. Centrifuge at 300 x g for 5 min at 4 °C, discard supernatant and resuspend in 1 ml PBS/BSA per donor mouse.
    14. Count cells, expecting a loss of 10-40% from the original cell count (Step B5) due to depletion and repeated wash steps.
    15. Confirm depletion of T cells by staining samples of pre- and post-depletion cells with fluorescent streptavidin and analysing by flow cytometry. Expect to see ~5% of lymphocytes staining positive in the pre-depletion sample, reduced to < 0.5% of lymphocytes in the post-depletion sample.

  3. Bone marrow transplantation (Day 0)
    1. Allow host mice 24 h recovery after the second injection of busulfan before transplantation with T-cell depleted donor bone marrow.
    2. Resuspend T-cell depleted bone marrow in PBS to a concentration of at least 50 million cells/ml.
    3. Deliver 200 μl (i.e., at least 10 million cells) to host mice by intravenous injection into the tail vein.

Data analysis

  1. At the desired timepoint(s) post-BMT, sacrifice chimeras and harvest organs of interest for analysis by flow cytometry. Organs and staining panels vary depending on the cell type of interest, but the inclusion of antibodies to both CD45.1 and CD45.2 is necessary in all staining panels to allow positive identification of cells of host (CD45.1+) or donor (CD45.2+) origin.
  2. By 6 weeks post-BMT, we typically see that the fraction of cells in the thymus that are donor-derived has stabilised at approximately 90%, and this is maintained up to one year post-BMT. The extent of stem cell replacement and therefore chimerism in progenitor populations varies between mice, reflecting variability in the ablation of host stem cells in the bone marrow. Thus the output of de novo generated T cells from the thymus is chimeric with respect to donor and host. To account for this, we measure the donor: host ratio within the thymus CD4+CD8+DP population of all mice as an accurate proxy for stem cell replacement in the bone marrow. With this estimate of stem cell chimerism, it is possible to estimate what fraction of any haematopoietic compartment has been replaced since the time of BMT, so called the fraction of ‘new’ cells, by the following formula:



    Since this calculation in effect normalises data against variability in stem cell chimerism, such estimates of ‘fraction new’ can be directly compared between individuals regardless of variability of stem cell engraftment following busulfan treatment (Figure 2).
  3. Timepoints chosen for analysis of chimeras depend upon the cell population of interest. For naïve CD4 and CD8 T cells, older host-derived cells are gradually replaced by new donor-derived cells until the donor:host ratio stabilises at approximately 30 weeks post-BMT (Figure 2D and Hogan et al., 2015). The replacement kinetics of memory CD4 T cell subsets are similar to naïve cells (Gossel et al., 2017), although the level of saturation varies by population, reflecting differences in underlying homeostatic mechanisms.


    Figure 2. Representative data analysis. A. Schematic of maturation stages for the T cell populations shown in Figures 2B-2D. B. Representative flow cytometry plots showing host (CD45.1+) and donor (CD45.2+) cells in chimeras 8 weeks post-BMT. Left panel shows thymocytes gated on CD4+CD8+ double positive cells; middle panel shows thymocytes gated on CD4+CD8- single positive cells; right panel shows splenocytes gated on naive (CD4+CD44lo) CD4 T cells. B. Fraction of donor cells in DP (left), CD4 SP (middle) and CD4 naive (right) populations at various times post-BMT. Each point represents one mouse. C. Fraction of ‘new’ cells in DP (left), CD4 SP (middle) and CD4 naive (right) populations at various times post-BMT. Each point represents one mouse. ‘Fraction new’ is calculated according to the formula (donor fraction in a population of interest)/(donor fraction in thymus DP).

Notes

  1. The busulfan dose used in this protocol (20 μg/g) was specifically tested in B6 CD45.1 mice and may require testing and titration in alternative mouse strains.
  2. Delivery of busulfan as two doses of 10 μg/g with 24 h recovery between injections is typically better tolerated by the mice than a single dose of 20 μg/g.
  3. The number of bone marrow cells injected can be varied from 10-20 million per mouse. However, doses lower than 10 million cells per mouse may result in a reduced level of chimerism.

Recipes

  1. PBS/BSA
    1. Supplement Dulbecco’s PBS with 1% BSA
    2. Keep sterile and store at 4 °C for up to 1 month

Acknowledgments

This protocol was developed for two published studies (Hogan et al., 2015; Gossel et al., 2017), both 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. Fry, T. J. and Mackall, C. L. (2005). Immune reconstitution following hematopoietic progenitor cell transplantation: challenges for the future. Bone Marrow Transplant 35 Suppl 1: S53-57.
  2. Goldrath, A. W., Luckey, C. J., Park, R., Benoist, C. and Mathis, D. (2004). The molecular program induced in T cells undergoing homeostatic proliferation. Proc Natl Acad Sci U S A 101(48): 16885-16890.
  3. 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.
  4. Hogan, T., Gossel, G., Yates, A. J. and Seddon, B. (2015). Temporal fate mapping reveals age-linked heterogeneity in naive T lymphocytes in mice. Proc Natl Acad Sci U S A 112(50): E6917-6926.
  5. Hsieh, M. M., Langemeijer, S., Wynter, A., Phang, O. A., Kang, E. M. and Tisdale, J. F. (2007). Low-dose parenteral busulfan provides an extended window for the infusion of hematopoietic stem cells in murine hosts. Exp Hematol 35(9): 1415-1420.
  6. Westerhof, G. R., Ploemacher, R. E., Boudewijn, A., Blokland, I., Dillingh, J. H., McGown, A. T., Hadfield, J. A., Dawson, M. J. and Down, J. D. (2000). Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients. Cancer Res 60(19): 5470-8.

简介

该方案被开发用于生成嵌合小鼠,其中T淋巴细胞可以根据同基因标志物表达的年龄进行分层。 白藜芦醇调理药物用于消融宿主造血干细胞,同时使外周免疫系统保持完整。 接受白消安治疗的是骨髓移植(BMT),T细胞耗竭的供体骨髓携带与宿主小鼠(CD45.1)不同的同基因标记(CD45.2)。 因此可以通过在感兴趣的群体内随时间测量CD45.2 +细胞的比例来追踪新的细胞生成后BMT(Hogan等人,2015; Gossel ,2017)。

【背景】骨髓嵌合体是研究免疫系统发育和功能的有价值的工具。典型地,通过照射宿主小鼠然后用供体骨髓移植产生嵌合体。辐射对造血系统造成相当大的损害,并且完全免疫重建在移植后延迟数周至数月(Fry和Mackall,2005)。由此产生的淋巴细胞减少期促使幼稚T细胞的自发性增殖和获得类似记忆的表型(Goldrath等人,2004)。为了避免辐射引起的免疫稳态的紊乱,我们把注意力转向调理药物白消安。白消安是一种烷化剂,对造血干细胞(HSC)有毒性,但不会耗竭循环淋巴细胞(Westerhof等人,2000; Hsieh等人,2007) 。在白消安调理和骨髓移植(BMT)之后,嵌合体与未处理的对照没有区别:它们具有正常数量的幼稚和记忆CD4和CD8T细胞,并且这些细胞表达增殖标记Ki67的正常水平(Hogan等人。,2015; Gossel et。,2017)。因此,白消安调节允许诱导嵌合同时保持外周免疫稳态。通过使用带有同源标记的供体骨髓,可以通过流式细胞术来区分宿主或供体来源的细胞。尽管HSC置换不是绝对的,但是供体来源细胞的出现可以被用作自BMT以来的造血系统发育的准确代表,从而允许群体内的细胞根据年龄分层标记表达。

关键字:T细胞, T细胞稳态, 骨髓嵌合体, 白消安, 时间命运映射

材料和试剂

  1. 5毫升注射器(BD,目录号:302187)
  2. 25 G针(Terumo,产品目录号:GS-351)
  3. 胰岛素注射器(B.BraunMelsungen,目录号:9151125)
  4. 培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150288)
  5. 70微米细胞过滤器(Corning,Falcon ,目录号:352350)
  6. 50毫升管(Corning,Falcon ,目录号:352070)
  7. 带有盖子的FACS管(Corning,Falcon ,目录号:352058)
  8. 宿主小鼠:B6 CD45.1成年雌性(THE JACKSON LABORATORY,目录号:002014)
  9. 供体小鼠:C57BL / 6 CD45.2成年雌性(THE JACKSON LABORATORY,目录号:000664)
    注意:一个供体通常为两名受体提供足够的T细胞耗竭的骨髓。
  10. Busilvex 6 mg / ml白消安注射液(Pierre Fabre)
  11. 生物素化的抗TCRβ(Thermo Fisher Scientific,eBioscience TM,目录号:13-5961)
  12. 生物素化的抗CD3ε(Thermo Fisher Scientific,eBioscience TM,产品目录号:13-0031)
  13. Dynabeads M-280链霉亲和素(Thermo Fisher Scientific,Invitrogen TM,目录号:11206D)
  14. 荧光抗生蛋白链菌素(如Streptavidin APC,BioLegend,目录号:405207)
  15. 对CD45.1的荧光抗体(例如抗CD45.1 Brilliant Violet 650,BioLegend,目录号:110736)
  16. 对CD45.2的荧光抗体(例如,抗CD45.2 PE-Dazzle 594,BioLegend,目录号:109846)
  17. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190)
  18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  19. PBS / BSA(见食谱)

设备

  1. 解剖工具(锋利的剪刀和镊子)
  2. 冷藏台式离心机
  3. EasySep™磁铁(STEMCELL Technologies,目录号:18000)
  4. 管转子
  5. 流式细胞仪

程序

注意:这个程序中的步骤总结在图1中。所有的动物程序都是按照英国内政部的规定进行的。


图1.白消安嵌合体小鼠的产生。该示意图总结了该方案用于产生白消安嵌合体小鼠的主要步骤。用来自CD45.2 +供体小鼠的骨髓细胞用CD3和TCRb的生物素化抗体标记,随后用链霉亲和素包被的磁珠标记。使用磁体将标记的细胞固定,从而收集未结合的部分。然后将T细胞耗尽的骨髓细胞注射到经白消毒剂调理的CD45.1 +宿主小鼠中。

  1. 白消安处理宿主CD45.1小鼠(第-2天和第-1天)
    1. 通过用无菌PBS稀释Busilvex(6毫克/毫升白消安)制备1毫克/毫升白消安的新鲜的工作溶液。
      注意:这必须每天新鲜准备,不能存储。
    2. 在每次注射之前,对每只小鼠分别进行标记和称重,并计算输送剂量为10μg/ g白消安的所需工作溶液的合适体积,每次10μl的1mg / ml工作溶液克体重。
    3. 第二天:腹腔注射适量白消安。

    4. 注射后允许宿主小鼠恢复24小时
    5. 第一天:重复步骤A1-A4,再次注射白消安。因此,每只小鼠中白消安的最终剂量为20μg/ g,相隔24 h两次剂量为10μg/ g。
      注意:白消安注射后小鼠保持健康和警觉。白消安处理后24h内HSC消耗(Westerhof et al。,2000)。

  2. 制备T细胞耗尽的供体CD45.2骨髓(第0天)
    注:细胞悬液可以在工作台上制备;不需要层流罩。

    1. 安乐死的捐助小鼠颈椎脱臼,并从两个后腿删除股骨和胫骨。
    2. 使用配有25 G针头的5 ml注射器,用PBS / BSA(参见食谱)冲洗每根骨头,在培养皿中收集骨髓。

    3. 过滤骨髓通过一个70微米细胞过滤器到50毫升管
    4. 在300℃下离心5分钟5分钟,弃去上清液并重悬于每只供体小鼠1ml PBS / BSA中。
    5. 计数细胞(预计每个供体小鼠可恢复约4000万个细胞),并调整细胞悬液的体积至20-40百万个细胞/ ml。
    6. 加入抗TCRβ和抗CD3ε生物素标记的抗体至终浓度为1μg/ ml,分别在4°C旋转孵育30分钟。
    7. 在4℃下300×g离心5分钟,弃去上清液并以浓度为20-40百万细胞/ ml重悬于PBS / BSA中。
    8. 将细胞悬液转移至5ml带有盖子的FACS管中。
      如果需要的话,使用多个管,每个管的体积为3-4毫升
    9. 添加抗生蛋白链菌素Dynabeads约1:1的珠子:细胞比例,并在4°C旋转孵育30分钟。
    10. 将带有细胞悬液的珠子管插入Easysep磁铁,在冰上孵育2分钟。
    11. 将含有未结合部分细胞的上清液收集到新鲜的试管中。
    12. 用收集的未结合的细胞部分重复步骤B10-B11,以确保从细胞悬液中除去所有珠子。
    13. 在300℃下离心5分钟5分钟,弃去上清液并重悬于每只供体小鼠1ml PBS / BSA中。
    14. 计数细胞,由于耗尽和重复洗涤步骤,预计从原始细胞计数(步骤B5)损失10-40%。
    15. 通过用荧光抗生蛋白链菌素染色消耗前和消减后细胞的样品并通过流式细胞术分析来确认T细胞的消耗。预期在去除前样品中约5%的淋巴细胞呈阳性染色,消耗后样品中0.5%的淋巴细胞。

  3. 骨髓移植(第0天)
    1. 在T细胞耗竭的供体骨髓移植之前,允许宿主小鼠在第二次注射白消安之后恢复24小时。

    2. 在PBS中重新悬浮T细胞耗尽的骨髓浓度至少5000万个细胞/毫升。
    3. 通过静脉注射到尾静脉中提供200μl(即,至少1000万个细胞)至宿主小鼠。

数据分析

  1. 在BMT后的期望时间点,牺牲嵌合体并收获感兴趣的器官用于通过流式细胞术进行分析。器官和染色面板根据感兴趣的细胞类型而变化,但是在所有染色面板中包含针对CD45.1和CD45.2的抗体是必需的,以允许阳性鉴定宿主细胞(CD45.1 + / sup>)或供体(CD45.2 + )来源。
  2. 在BMT后6周,我们通常可以看到,供体来源的胸腺细胞分数稳定在大约90%,这种情况在BMT后一年内维持不变。干细胞替代的程度和因此祖细胞群体中的嵌合状态在小鼠之间变化,这反映了骨髓中宿主干细胞的消融的变化。因此从胸腺产生的从头产生的T细胞相对于供体和宿主是嵌合的。为了解释这一点,我们测量了所有小鼠的胸腺CD4 + CD8 + DP群体内的供体:宿主比例作为骨髓中干细胞替代的准确代表。有了这个干细胞嵌合体的估计,有可能估计自BMT时间以来,已经更换了任何造血隔室的部分,所谓的“新”细胞的部分,按照下面的公式:



    由于该计算有效地将数据与干细胞嵌合体的变异性进行归一化,所以可以直接比较个体之间的“新的分数”的这种估计值,而不管白消安治疗后的干细胞植入的变化(图2)。
  3. 选择用于分析嵌合体的时间点取决于感兴趣的细胞群体。对于幼稚的CD4和CD8T细胞,更老的宿主来源的细胞逐渐被新的供体来源的细胞替代,直到供体:宿主比率在BMT后大约30周稳定(图2D和Hogan等人,2015)。记忆CD4 T细胞亚群的替代动力学与初始细胞类似(Gossel等人,2017),尽管饱和度水平因人群而异,反映了潜在的稳态机制的差异。


    图2.代表性的数据分析。 :一种。图2B-2D所示的T细胞群的成熟阶段示意图。 B.代表性流式细胞术图显示BMT后8周嵌合体中的宿主(CD45.1 + +)和供体(CD45.2 +)细胞。左图显示在CD4 + CD8 +双阳性细胞上门控的胸腺细胞;中图显示在CD4 + CD8 - +单阳性细胞上门控的胸腺细胞;右图显示在天真(CD4 + CD44 +)CD4 T细胞上门控的脾细胞。 B.在BMT后不同时间在DP(左),CD4 SP(中)和CD4幼稚(右)群体中的供体细胞的分数。每个点代表一只老鼠。 C.在BMT后不同时间在DP(左),CD4 SP(中)和CD4幼稚(右)群体中的“新”细胞的分数。每个点代表一只老鼠。根据公式(感兴趣的人群中的供体分数)/(胸腺DP中的供体分数)计算“分数新”。

笔记

  1. 在该方案中使用的白消安剂量(20μg/ g)在B6 CD45.1小鼠中被特异性测试,并且可能需要在替代的小鼠品系中进行测试和滴定。
  2. 在注射之间24小时恢复时,以10μg/ g的两个剂量递送白消安通常比单剂量20μg/ g更能被小鼠耐受。
  3. 注射的骨髓细胞数量可以从每只小鼠10-20万个变化。然而,低于每个小鼠1000万个细胞的剂量可能导致嵌合体水平降低。

食谱

  1. PBS / BSA
    1. 补充Dulbecco的PBS与1%BSA
    2. 保持无菌状态并在4°C储存长达1个月

致谢

该协议是为国家卫生研究院(R01 AI093870)支持的两项已发表的研究(Hogan等人,2015; Gossel等人,2017)开发的,和医学研究委员会(MC-PC-13055)。作者声明不存在利益冲突或利益冲突。

参考

  1. Fry,T.J。和Mackall,C.L。(2005)。 造血祖细胞移植后的免疫重建:对未来的挑战 Bone骨髓移植 35 Suppl 1:S53-57。
  2. Goldrath,A.W.,Luckey,C.J.,Park,R.,Benoist,C.和Mathis,D。(2004)。 在正在经历稳态增殖的T细胞中诱导的分子程序 Proc Natl Acad Sci USA 101(48):16885-16890。
  3. 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.
  4. Hogan,T.,Gossel,G.,Yates,A.J。和Seddon,B。(2015)。 时间的命运映射揭示了幼稚T淋巴细胞在年龄上的异质性。 > Proc Natl Acad Sci USA 112(50):E6917-6926。
  5. Hsieh,M.M.,Langemeijer,S.,Wynter,A.,Phang,O.A。,Kang,E.M。和Tisdale,J.F。(2007)。 低剂量肠外白消安提供了在鼠宿主中输注造血干细胞的延长窗口。 / Exp> Hematol 35(9):1415-1420。
  6. Westerhof,G.R.,Ploemacher,R.E.,Boudewijn,A.,Blokland,I.,Dillingh,J.H.,McGown,A.T.,Hadfield,J.A。,Dawson,M.J。和Down,J.D。(2000)。 比较不同的白消安类似物对造血干细胞的耗竭和促进小鼠骨髓供体型嵌合骨髓移植受者。 癌症研究 60(19):5470-8。
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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). Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics. Bio-protocol 7(24): e2650. DOI: 10.21769/BioProtoc.2650.
  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.
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