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Limiting Dilution Assays to Determine Frequencies of Lymphohematopoietic Progenitors
有限稀释法测定淋巴造血祖细胞的比例

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Abstract

This protocol is useful to determine the frequencies of lymphohematopoietic progenitors in tested samples. To effectively support the growth and differentiation of primitive lymphohematopoietic progenitors, complex signals from stromal cells are important. Several stromal cell lines are known to support both lymphoid and myeloid cells simultaneously in mouse. In this protocol, we introduce two stromal co-culture systems for murine lymphohematopoietic progenitors and their application for limiting dilution assays.

Materials and Reagents

  1. Stromal cells (MS5 cells or OP9 cells)
  2. Growth factors
    a. rm SCF (10 ng/ml) (e.g. R&D systems, catalog number: 455-MC-010 )
    b. rm Flt3-ligand (20 ng/ml) (e.g. R&D systems, catalog number: 427-FL-025 )
    c. rm IL-7 (1 ng/ml) (e.g. R&D systems, catalog number: 407-ML-005 )
  3. Antibodies
    a. Anti-mouse Mac1 (BD Biosciences, catalog number: 557396 )
    b. Gr1 (BD Biosciences, catalog number: 553128 )
    c. CD19 (BD Biosciences, catalog number: 557399 )
    d. CD45R/B200 (BD Biosciences, catalog number: 553092 )
    e. CD45 (BD Biosciences, catalog number: 550994 )
  4. Trypsin/EDTA solution (Nacalai Tesque, catalog number: 35554-64 )
  5. EDTA solution (Nacalai Tesque, catalog number: 14367-74 )
  6. Alpha Modification of Eagle’s Medium (Corning, cellgro®, catalog number: 50-012 )
  7. Minimum Essential Medium Alpha Medium (Gibco®, catalog number: 41061-029 )
  8. FCS (MP Biomedicals, catalog number: 2916754 )
  9. L-glutamine (Corning, cellgro®, catalog number: 61-030 )
  10. Penicillin-Streptomycin (Nacalai Tesque, catalog number: 26253-84 )
  11. MS-5 growth media (see Recipes)
  12. OP-9 growth media (see Recipes)

Equipment

  1. Cell sorting machines (BD Biosciences, FACSAriaTM) with the automated cell deposition system
  2. 96-well flat bottom plates
  3. 10 cm plastic dishes
  4. CO2 incubators
  5. Flow cytometry system

Procedure

  1. Grow stromal cells to a sub-confluent state in 10 cm-dishes. Use MS-5 growth media to grow MS-5 stromal cells. Use OP-9 growth media to grow OP-9 stromal cells.
  2. Wash the stromal cells twice with PBS and trypsinize with trypsin/EDTA solution at 37 °C for 5 min. Dissociate the cells by pipetting. After centrifugation at 300 x g for 5 min, re-suspend the cells in the growth medium. Seed half of the cells from a 10 cm dish into one 96-well plate on a day before the start of co-culture. The density and volume of stromal cells are 40~50 cells/ml and 50 ml/well when they are seeded into 96-well plates.
  3. Prepare testing lymphohematopoietic progenitors. (The testing progenitors should differ according to the aim of each experiment.) Put the progenitors with the different densities (for example 1, 2, 4, 8, 16, 32, 64 cells per well) onto the prepared stromal layers by using a cell-sorting machine. Culture the progenitors on stromal cells in alpha Modification of Eagle’s Medium supplemented with 10% FCS, rmSCF (10 ng/ml), rmFlt3-ligand (20 ng/ml), and rmIL7 (1 ng/ml).
  4. Feed the cultures twice a week by removing half of the medium and replacing it with fresh medium. Cytokines should be freshly added with each feeding.
  5. At 10 days of culture, inspect the wells for the presence of hematopoietic clones. Positive wells are harvested and analyzed by flow cytometry for the presence of CD45+ hematopoietic cells, CD45R/B220+ CD19+ Mac1- (and/or Gr1-) B-lineage cells, and Mac1+ (and/or Gr1+) myeloid-lineage cells (Figure 1).


    Figure 1. Shown above is a representative profile of flow cytometry showing the expression of Mac1 and CD19 on recovered CD45+ cells (from Yokota et al., 2009)

  6. Determine the frequencies of progenitors by calculating with linear regression analysis on the basis of Poisson distribution. The frequencies are given as the reciprocal of the concentration of test cells that result in 37% negative cultures. See Figure 2 for an example.


    Figure 2. Semilogarithmic plots for hematopoietic cells and B lineage cells are made of % of negative cultures as a function of the dose of input ESAM-/Lo or ESAM+ progenitors in each well. One in 2.1 ESAMHi cells and 1 in 3.5 ESAM-/Lo cells gave rise to CD45+ blood cells, indicating that both populations are potent sources of hematopoietic progenitors (left). However, while 1 in 8 ESAMHi cells gave rise to CD19+ B lineage cells, only 1 in 125 ESAM-/Lo cells produced B lineage cells under these conditions (right) (from Yokota et al., 2009).

Recipes

  1. MS-5 growth media
    Alpha Modification of Eagle’s Medium
    10% FCS
    2 mM L-glutamine
    Penicillin-Streptomycin
  2. OP-9 growth media
    Minimum Essential Medium Alpha Medium
    20% FCS
    Penicillin-Streptomycin

Acknowledgments

This protocol was adapted from our previously published papers Satoh et al. (2013) and Yokota et al. (2009). The representative data shown in the protocol was adapted from Yokota et al. (2009).

References

  1. Satoh, Y., Yokota, T., Sudo, T., Kondo, M., Lai, A., Kincade, P. W., Kouro, T., Iida, R., Kokame, K., Miyata, T., Habuchi, Y., Matsui, K., Tanaka, H., Matsumura, I., Oritani, K., Kohwi-Shigematsu, T. and Kanakura, Y. (2013). The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages. Immunity 38(6): 1105-1115.
  2. Yokota, T., Oritani, K., Butz, S., Kokame, K., Kincade, P. W., Miyata, T., Vestweber, D. and Kanakura, Y. (2009). The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice. Blood 113(13): 2914-2923.

简介

这个协议是有用的确定淋巴造血祖细胞在测试样品中的频率。 为了有效支持原始淋巴造血祖细胞的生长和分化,来自基质细胞的复合信号是重要的。 已知几种基质细胞系在小鼠中同时支持淋巴和骨髓细胞。 在这个协议,我们介绍两个基质共培养系统鼠淋巴造血祖细胞及其应用限制稀释测定。

材料和试剂

  1. 基质细胞(MS5细胞或OP9细胞)
  2. 生长因子
    一个。 rm SCF(10ng/ml)(例如,R& D systems,目录号:455-MC-010)。
    b。 rm Flt3-配体(20ng/ml)(例如R& D systems,目录号:427-FL-025)
    C。 IL-7(1ng/ml)(例如R& D systems,目录号:407-ML-005)。
  3. 抗体
    一个。 抗小鼠Mac1(BD Biosciences,目录号:557396)
    b。 Gr1(BD Biosciences,目录号:553128) C。 CD19(BD Biosciences,目录号:557399) d。 CD45R/B200(BD Biosciences,目录号:553092) e。 CD45(BD Biosciences,目录号:550994)
  4. 胰蛋白酶/EDTA溶液(Nacalai Tesque,目录号:35554-64)
  5. EDTA溶液(Nacalai Tesque,目录号:14367-74)
  6. Eagle培养基的Alpha修饰(Corning,cellgro ,目录号:50-012)
  7. 最小必需培养基α培养基(目录号:41061-029)
  8. FCS(MP Biomedicals,目录号:2916754)
  9. L-谷氨酰胺(Corning,cellgro ,目录号:61-030)
  10. 青霉素 - 链霉素(Nacalai Tesque,目录号:26253-84)
  11. MS-5生长培养基(参见配方)
  12. OP-9生长培养基(参见配方)

设备

  1. 具有自动细胞沉积系统的细胞分选机(BD Biosciences,FACSAria TM
  2. 96孔平底板
  3. 10厘米塑料碗
  4. CO <2>孵化器
  5. 流式细胞术系统

程序

  1. 在10厘米的培养皿中生长基质细胞至亚汇合状态。 使用MS-5生长培养基培养MS-5基质细胞。 使用OP-9生长培养基生长OP-9基质细胞。
  2. 用PBS洗涤基质细胞两次,并用胰蛋白酶/EDTA溶液在37℃下胰蛋白酶消化5分钟。通过吸移分离细胞。在300×g离心5分钟后,将细胞重新悬浮在生长培养基中。在开始共培养前一天将一半来自10cm培养皿的细胞接种到一个96孔板中。当将其接种到96孔板中时,基质细胞的密度和体积为40〜50细胞/ml和50 ml /孔。
  3. 准备测试淋巴母细胞祖细胞。 (根据每个实验的目的,测试祖细胞应该不同。)将具有不同密度的祖细胞(例如每个孔1,2,4,8,16,32,64个细胞)置于制备的基质层上,使用细胞分选机。在补充有10%FCS,rmSCF(10ng/ml),rmFlt3-配体(20ng/ml)和rmIL7(1ng/ml)的Eagle's培养基的α修饰中的基质细胞上培养祖细胞。
  4. 通过去除一半培养基并用新鲜培养基替换来每周喂养两次培养物。每次喂养时应新鲜加入细胞因子。
  5. 在培养10天时,检查孔中是否存在造血克隆。收获阳性孔,并通过流式细胞术分析CD45 +/+造血细胞,CD45R/B220 +和CD19 +/+ Mac1阳性细胞的存在。 (和/或Gr1 - )B-谱系细胞和Mac1 + (和/或Gr1 + )髓系细胞(图1)。


    图1.上面显示的是流式细胞术的代表性图谱,显示Mac1和CD19在恢复的CD45 + 细胞上的表达横田 等 al。 ,2009)

  6. 通过基于泊松分布的线性回归分析计算确定祖细胞的频率。频率作为导致37%阴性培养物的测试细胞浓度的倒数给出。参见图2的示例。


    图2.造血细胞和B谱系细胞的半对数图由阴性培养物的%作为输入ESAM 剂量的函数, sup> 细胞和3.5个ESAM sup/Lo细胞中的1个产生CD45 +血细胞,表明两个群体都是造血祖细胞的有效来源(左)。然而,尽管8个ESAM sup细胞中有1个产生CD19 sup + B谱系细胞,但在125个ESAM sup/Lo细胞中只有1个产生B细胞在这些条件下(右)(来自Yokota et al。,2009)的谱系细胞。

食谱

  1. MS-5生长培养基
    Eagle氏培养基的Alpha修饰
    10%FCS
    2mM L-谷氨酰胺 青霉素 - 链霉素
  2. OP-9生长培养基
    最低必需培养基中度
    20%FCS
    青霉素 - 链霉素

致谢

该协议改编自我们先前发表的论文Satoh等人(2013)和Yokota等人(2009)。 方案中所示的代表性数据来自Yokota等人(2009)。

参考文献

  1. Satoh,Y.,Yokota,T.,Sudo,T.,Kondo,M.,Lai,A.,Kincade,PW,Kouro,T.,Iida,R.,Kokame,K.,Miyata,T.,Habuchi ,Y.,Matsui,K.,Tanaka,H.,Matsumura,I.,Oritani,K.,Kohwi-Shigematsu,T.and Kanakura,Y。 Satb1蛋白指导造血干细胞向淋巴谱系分化。 免疫 38(6):1105-1115。
  2. Yokota,T.,Oritani,K.,Butz,S.,Kokame,K.,Kincade,P.W.,Miyata,T.,Vestweber,D.and Kanakura,Y。 内皮抗原ESAM在小鼠的整个生命中标记原始造血祖细胞。血液 113(13):2914-2923。
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引用:Yokota, T. and Satoh, Y. (2014). Limiting Dilution Assays to Determine Frequencies of Lymphohematopoietic Progenitors. Bio-protocol 4(4): e1045. DOI: 10.21769/BioProtoc.1045.
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