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In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells
小鼠造血干细胞的体内BrdU掺入标记试验   

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本实验方案简略版
Stem Cells
Jun 2013

Abstract

Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure of cell proliferation in adult murine hematopioetic stem cells (HSCs). Specifically, BrdU incorporation was analyzed for long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+) population.

Materials and Reagents

  1. Mouse
  2. BrdU
  3. RPMI1640
  4. Fetal Bovine Serum (FBS)
  5. Potassium bicarbonate
  6. Ammonium chloride
  7. EDTA
  8. BSA
  9. Lineage Cell Depletion Kit (including Biotin-Antibody Cocktail and Anti-Biotin MicroBeads) (Miltenyi Biotec, catalog number: 130-090-858 )
  10. BD Cytofix/Cytoperm Buffer (Becton, Dickinson and Company, catalog number: 554714 )
  11. BD Perm/Wash Buffer
  12. DNase (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619 )
  13. DPBS without calcium, magnesium (diluted from 10x DPBS) (Hyclone, catalog number: SH 30258.01 )
  14. Antibodies:
    PE-conjugated-Sca-I (Becton, Dickinson and Company, catalog number: 553336 )
    APC-conjugated-c-Kit (Becton, Dickinson and Company, catalog number: 553356 )
    PerCP-eFluor 710-conjugated-CD135 (eBioscience, catalog number: 46-1351-80 )
    eFluor 450-conjugated CD34 (eBioscience, catalog number: 48-0341-80 )
    BrdU-FITC (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619)
  15. Buffer A (see Recipes)
  16. Red blood cell lysis buffer (see Recipes)
  17. Staining buffer (see Recipes)

Equipment

  1. Small scissors and forceps
  2. 60 mm tissue culture dish
  3. 23 G needle
  4. 3 cc syringe
  5. 15 ml centrifuge tube
  6. Cell strainer (Becton, Dickinson and Company, catalog number: 352340 )
  7. Hemacytometer
  8. MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 )
  9. MiniMASC separator (Miltenyi Biotec, catalog number: 130-042-102 )
  10. Centrifuge
  11. BD LSRFortessa Analytical Flow Cytometer

Procedure

  1. Animal injection  
    i.p. Injection of BrdU (10 mg/ml) 100 μl to 8-12 weeks old mouse (50 μg/g BW, so 5 μl/g of BW), after 6 h, inject the second dose. 2 h post 2nd injection, euthanize the mice using CO2 method followed by cervical dislocation, obtain the bone marrow (BM) cells from 2 tibias and 2 femurs. Two injections ensure that BrdU can incorporate to DNA of both slow and quick turnover cells.   
  2. Obtain total bone marrow (BM) cells
    1. Use small scissors and forceps, dissect out femurs and tibias from mice and place them in a 60 mm tissue culture dish containing 6 ml ice-cold RPMI1640 with 5% heat inactivated FBS. Use Kimwipe tissue to remove muscle and other tissues. Cut off both ends of each bone shaft in the dish.
    2. Connect the end of the bone with 23 G needle on 3 cc syringe, flush out bone marrow with RPMI1640 with 5% heat inactivated FBS into the dish. Disaggregate bone marrow tissues by repeated aspirations using the same needle. Transfer the cell suspension to 15 ml centrifuge tube.
    3. Spin down the cells at 350 x g for 5 min at room temperature, remove the supernatant, resuspend the cells in 1 ml of room temperature red blood cell lysis buffer and incubate at room temperature for 5 min, then add 5-10 ml of RPMI 1640 with 5% heat inactivated FBS.
    4. Pass the cells through a cell strainer. Collect the flow through to a new tube. Take an aliquot and count the cells in a hemacytometer. Spin down at 350 x g for 5 min at room temperature. Remove the supernatant; the cell pellet should not contain any red color. Disaggregate the cell pellet and wash the cells one time with buffer A, spin down at 350 x g for 5 min at room temperature.
  3. Lineage cell staining (follow the mouse Lineage Cell Depletion Kit)  
    1. Resuspend the total BM cells in buffer A (40 μl/107 cells).
    2. Add Biotin-Antibody Cocktail (10 μl/107 cells) to stain the lineage differentiated cells. Cocktail of biotin-conjugated monoclonal antibodies contains anti-CD5, anti-CD45R(B220), anti-CD11b, anti-Gr-1(Ly-6G/C), anti-Neutrophil (7/4) and anti-Ter-119.
    3. Mix well and incubate for 10 min at 4 °C.
    4. Add additional buffer A in media (30 μl/107 cells) then add Anti-Biotin MicroBeads (20 μl/107 cells, provided in Lineage Cell Depletion Kit).
    5. Mix well and incubate for 15 min at 4 °C.
    6. Wash cell by adding 2 ml of buffer A. Centrifuge at 300 x g for 10 min at room temperature.
    7. Remove the supernatant and resuspend the pellet in 0.5 ml of buffer A.
  4. Lineage depleation
    1. Place MACS MS column in MiniMASC separator.
    2. Prepare column by rinsing with 0.5 ml buffer A.
    3. Apply cell suspension onto the column. Allow the cells to pass through and collect flow through as Lin- fraction.
    4. Wash column 3 times with buffer A (0.5 ml/each), wash each time once the column reservoir is empty.
    5. Collect all the elute (Lin-) in one tube.
    6. Count the Lin- cells, aliquot 1.5 x 106 cells to a new tube.
    7. Add 2 times more staining buffer to the cell suspension.
    8. Centrifuge the cells at 350 x g for 5 min.
  5. Stain the Lin- cells with surface antigens:
    Test Total (μl) Percp-eFluor 710 eFluor 450 PE APC Staining
    buffer
    CD135 CD34 Sca-I
    CD117
    (c-kit)
    1 75 1 2 0.5 0.5 71
    1. Stain each test sample per 1.5 x 106 cells/75 μl buffer, make antibody mix as following, for more samples, increase antibody amount and staining buffer proportionally.
    2. Add 75 μl of antibody mix to the cell pellet. Incubate cells with antibodies for 15 minutes at room temperature (protected from light).
    3. Wash one time with staining buffer. Spin down for 5 minutes at 350 x g, and discard the supernatant.
  6. Fix and permeabilize the cells
    1. Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
    2. Incubate the cells for 15 to 30 minutes at room temperature or on ice.
    3. Wash the cells with 1 ml of 1x BD Perm/Wash Buffer (dilute the 10x buffer with deionized H2O). Centrifuge at 350 x g for 5 minutes at room temperature, and discard the supernatant.
  7. Enhance the permeabilization:  
    1. Resuspend the cells in 100 μl of BD Cytoperm Permeabilization Buffer Plus per tube. This reagent is specially formulated for the BrdU Flow kit and is used as a staining enhancer and secondary permeabilization reagent.
    2. Incubate the cells for 10 minutes on ice.
    3. Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
  8. Re-fix cells after secondary permeabilization:
    1. Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
    2. Incubate the cells for 5 minutes at room temperature or on ice.
    3. Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
  9. Treat with DNase to expose incorporated BrdU
    1. Resuspend the cells in 100 μl of diluted DNase (diluted to 300 μg/ml in DPBS) per tube, (i.e. 30 μg of DNase/106 cells).
    2. Incubate cells for 1 hour at 37 °C.
    3. Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
  10. BrdU intracellular antigens staining
    1. Make diluted BrdU antibody (1 μl to 50 μl/sample in BD Perm/Wash Buffer).
    2. Incubate the cells for 20 minutes at room temperature.
    3. Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 4c).
  11. Resuspend the cells in 0.3 ml of staining buffer and perform flow cytometry analysis. Samples can be stored overnight at 4 °C, protected from light, prior to analysis by flow cytometry.
  12. Flow cytometry was performed on BD LSRFortessa Analytical Flow Cytometer with the following gating strategy (Figure 1).


    Figure 1. Gating strategy to analyze BrdU incorporation in LT-HSCs. A. BM Lin- cells were labeled with PE-Scal-I and APC-c-Kit antibodies and analyzed by flow cytometry. B. Lin-/Scal-I+/c-Kit+ (LSK) cells were gated as showed in A and the LSK cells were further analyzed with eFluor 450-CD34 and PerCP-eFluor 710-CD135 staining. Long-term HSCs (LT-HSCs, shown as Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, shown as Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, shown as Lin-Sca-1+c-Kit+CD34+CD135+) were separated as indicated. C. BrdU incorporation was further analyzed for each cell population. Data shown are LT-HSCs population analyzed for BrdU staining.

Recipes

  1. Buffer A
    DPBS, pH 7.2 supplemented with 0.5% BSA and 2 mM EDTA
  2. Red blood cell lysis buffer
    155 mM potassium bicarbonate
    10 mM Ammonium chloride
    0.1 mM of EDTA, pH = 7.4
  3. Staining buffer
    DPBS, pH 7.2 supplememted with 0.5% BSA and 0.09% sodium azide

Acknowledgments

This protocol is adapted from An et al. (2013).

References

  1. An, N., Lin, Y. W., Mahajan, S., Kellner, J. N., Wang, Y., Li, Z., Kraft, A. S. and Kang, Y. (2013). Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells. Stem Cells 31(6): 1202-1212.   

简介

溴脱氧尿苷(BrdU)是在细胞周期的S期期间掺入DNA的胸苷类似物。 因此,BrdU掺入可用于定量在BrdU可用的时间段内处于S期的细胞的数量。 以下方案描述了体内BrdU掺入测定作为成体鼠造血干细胞(HSC)中细胞增殖的量度。 具体地,分析BrdU掺入的长期HSC(LT-HSC,Lin-Sca-1 + c-Kit + CD34 - CD135 - ),短期HSC(ST-HSC,Lin-Sca-1 + c-Kit + CD34 和多能祖细胞(MPP,Lin-Sca-1 + c-Kit + CD34 + > CD135 + )群体

材料和试剂

  1. 鼠标
  2. BrdU
  3. RPMI1640
  4. 胎牛血清(FBS)
  5. 碳酸氢钠
  6. 氯化铵
  7. EDTA
  8. BSA
  9. 谱系细胞消耗试剂盒(包括生物素 - 抗体混合物和抗生物素微珠)(Miltenyi Biotec,目录号:130-090-858)
  10. BD Cytofix/Cytoperm缓冲液(Becton,Dickinson and Company,目录号:554714)
  11. BD Perm/Wash Buffer
  12. DNase(包含在FITC BrdU Flow Kit中)(Becton,Dickinson and Company,目录号:559619)
  13. 无钙的DPBS,镁(从10×DPBS稀释)(Hyclone,目录号:SH 30258.01)
  14. 抗体:
    PE结合的Sca-I(Becton,Dickinson and Company,目录号:553336) APC结合的c-Kit(Becton,Dickinson and Company,目录号:553356) PerCP-eFluor 710-共轭-CD135(eBioscience,目录号:46-1351-80)
    eFluor 450-共轭CD34(eBioscience,目录号:48-0341-80) BrdU-FITC(包含在FITC BrdU Flow Kit中)(Becton,Dickinson and Company,目录号:559619)
  15. 缓冲区A(参见配方)
  16. 红细胞裂解缓冲液(见配方)
  17. 染色缓冲液(见配方)

设备

  1. 小剪刀和镊子
  2. 60 mm组织培养皿
  3. 23 G针
  4. 3毫升注射器
  5. 15ml离心管
  6. 细胞过滤器(Becton,Dickinson and Company,目录号:352340)
  7. 血细胞计数器
  8. MACS MS柱(Miltenyi Biotec,目录号:130-042-201)
  9. MiniMASC分离器(Miltenyi Biotec,目录号:130-042-102)
  10. 离心机
  11. BD LSRFortessa分析流式细胞仪

程序

  1. 动物注射
    腹膜内。 注射BrdU(10mg/ml)100μl至8-12周龄小鼠(50μg/g BW,因此5μl/g BW),6小时后,注射第二剂量。 在第2次注射后2小时,使用CO 2方法随后颈椎脱位使安乐死小鼠,从2个胫骨和2个股骨获得骨髓(BM)细胞。 两次注射确保BrdU可以并入慢和快速周转细胞的DNA。  
  2. 获得总骨髓(BM)细胞
    1. 使用小剪刀和镊子,从股骨和胫骨解剖 小鼠并将其放置在含有6ml的60mm组织培养皿中 冰冷的RPMI1640与5%热灭活的FBS。 使用Kimwipe组织 去除肌肉和其他组织。 切断每个骨轴的两端   菜。
    2. 连接骨头的末端与23 G针3   cc注射器,用5%热的RPMI1640冲洗出骨髓 灭活的FBS进入培养皿。 解聚骨髓组织 使用相同的针重复吸气。 转移细胞悬浮液   至15ml离心管
    3. 在室温下以350×g离心细胞5分钟,除去上清液,重悬 细胞在1ml室温红细胞裂解缓冲液中 在室温下孵育5分钟,然后加入5-10ml的RPMI 1640 用5%热灭活的FBS
    4. 使细胞通过细胞 过滤器。 收集流量通过一个新的管。 取等分试样和 计数血细胞计数器中的细胞。 在350摄氏度下旋转5分钟,持续5分钟 室内温度。 去除上清液; 细胞沉淀不应该 包含任何红色。 分解细胞沉淀并洗涤细胞 用缓冲液A一次,在室温下以350×g离心5分钟 温度。
  3. 谱系细胞染色(按照小鼠谱系细胞耗竭试剂盒)
    1. 将总BM细胞重悬在缓冲液A(40μl/10μL,细胞)中
    2. 加   生物素 - 抗体混合物(10μl/10 7个细胞)染色谱系 分化细胞。 生物素偶联单克隆抗体的鸡尾酒 抗体含有抗CD5,抗CD45R(B220),抗CD11b, 抗Gr-1(Ly-6G/C),抗中性粒细胞(7/4)和抗Ter-119。
    3. 充分混合并在4℃下孵育10分钟
    4. 在培养基中加入另外的缓冲液A(30μl/10μL细胞),然后加入抗生物素微珠(20μl/10μL细胞,在Lineage Cell Depletion Kit中提供)。
    5. 充分混匀,在4℃下孵育15分钟
    6. 通过加入2ml缓冲液A洗涤细胞。在室温下以300×g离心10分钟。
    7. 除去上清液并将沉淀重悬在0.5ml缓冲液A中。
  4. 谱系衰竭
    1. 将MACS MS柱置于MiniMASC分离器中。
    2. 通过用0.5ml缓冲液A冲洗来制备柱
    3. 将细胞悬液应用于色谱柱。 允许细胞通过并收集流动通过作为线性
    4. 用缓冲液A洗涤柱3次(每次0.5ml),每次一次柱储存器为空时洗涤
    5. 在一个管中收集所有洗脱液(Lin-)。
    6. 计数Lin-细胞,将1.5×10 6个细胞等分到新管
    7. 向细胞悬液中加入2倍以上的染色缓冲液
    8. 离心细胞在350×g 5分钟。
  5. 用表面抗原染色Lincell:
    测试 总 (μl) Percp-eFluor 710 eFluor 450 PE APC 染色
    缓冲
    CD135 CD34 Sca-I
    CD117
    (c-kit)
    1 75 1 2 0.5 0.5 71
    1. 将每个测试样品每1.5×10 6个细胞/75μl缓冲液染色,对于更多样品,制备如下的抗体混合物,按比例增加抗体量和染色缓冲液。
    2. 加入75微升抗体混合物的细胞沉淀。 在室温(避光保存)下用抗体孵育细胞15分钟
    3. 用染色缓冲液洗涤一次。 在350×g下旋转5分钟,并弃去上清液。
  6. 固定和透化细胞
    1. 将细胞重悬在每管100μl的BD Cytofix/Cytoperm缓冲液中
    2. 在室温或冰上孵育细胞15至30分钟
    3. 用1ml 1×BD Perm/Wash缓冲液(用去离子H 2 O稀释10×缓冲液)洗涤细胞。 在室温下以350xg离心5分钟,弃去上清液。
  7. 增强透化:
    1. 重悬细胞在100微升BD Cytoperm渗透缓冲液Plus每管。 该试剂专为BrdU Flow试剂盒配制,用作染色增强剂和二次透化试剂。
    2. 在冰上孵育细胞10分钟。
    3. 在1ml的1x BD Perm/Wash Buffer中清洗细胞(如步骤5c)
  8. 在二次透化后重新固定细胞:
    1. 将细胞重悬在每管100μl的BD Cytofix/Cytoperm缓冲液中
    2. 在室温或冰上孵育细胞5分钟。
    3. 在1ml的1x BD Perm/Wash Buffer中清洗细胞(如步骤5c)
  9. 用DNase处理以暴露掺入的BrdU
    1. 将细胞重悬于100μl稀释的DNase(在DPBS中稀释至300μg/ml,在每管中)( 。30μgDNase/10 cells)。
    2. 在37℃下孵育细胞1小时
    3. 在1ml的1x BD Perm/Wash Buffer中清洗细胞(如步骤5c)
  10. BrdU细胞内抗原染色
    1. 制备稀释的BrdU抗体(1μl至50μl/样品在BD Perm/Wash Buffer中)
    2. 在室温下孵育细胞20分钟。
    3. 洗涤细胞在1毫升1x BD Perm/Wash缓冲液(如步骤4c)。
  11. 将细胞悬浮在0.3ml染色缓冲液中,进行流式细胞术分析。 样品可以在4℃下避光保存过夜,然后通过流式细胞术分析
  12. 流式细胞术在BD LSRFortessa分析流式细胞仪上进行,具有以下门控策略(图1)。


    图1.用于分析LT-HSC中BrdU掺入的门控策略 A.用PE-Scal-I和APC-c-Kit抗体标记BM Lin-细胞,并通过流式细胞术分析。如A中所示门控B.Linc - /Scal-I + /c-Kit + (LSK)细胞,LSK细胞用eFluor 450-CD34和PerCP-eFluor 710-CD135染色进一步分析。长期HSC(LT-HSC,如Lin - Sca-1 + c-Kit + CD34 - CD135 - ),短期HSC(ST-和多能祖细胞(MPP,显示于图1中),其中所述多能祖细胞包含表达CD34作为Lin - Sca-1 + c-Kit + CD34 + CD135 + )如所示分离。进一步分析每个细胞群的BrdU掺入。显示的数据是针对BrdU染色分析的LT-HSCs群体

食谱

  1. 缓冲区A
    DPBS,pH 7.2,补充有0.5%BSA和2mM EDTA
  2. 红细胞裂解缓冲液
    155 mM碳酸氢钠 10mM氯化铵
    0.1mM EDTA,pH = 7.4
  3. 染色缓冲区
    DPBS,pH 7.2用0.5%BSA和0.09%叠氮化钠补充

致谢

该协议改编自An (2013)。

参考文献

  1. An,N.,Lin,Y. W.,Mahajan,S.,Kellner,J. N.,Wang,Y.,Li,Z.,Kraft,A.S.and Kang,Y。 Pim1丝氨酸/苏氨酸激酶调节鼠造血干细胞的数量和功能 < em> Stem Cells 31(6):1202-1212。   
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:An, N. and Kang, Y. (2013). In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells. Bio-protocol 3(21): e960. DOI: 10.21769/BioProtoc.960.
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