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Competitive Bone-marrow Transplantations
竞争性骨髓移植   

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Abstract

Competitive bone marrow transplantation assay measures multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. Thus this assay is routinely used to determine haematopoietic stem and progenitor cells (HSPCs) functionality in vivo. The principle of the method is to transplant bone marrow donor cells (derived from transgenic mice of choice) on C57BL6 background together with normal competitor bone marrow. In order to distinguish donor from competitor cells upon transplantation, usually competitor mice are congenic and carry the differential B cell antigen originally designated Ly5.1 and CD45.1.
A typical competitive bone marrow transplantation experiment will contain two transplantation groups, donor (transgenic mice of choice and their controls) are transplanted in competition with normal competitors and engraftment efficiency is evaluated in both blood and bone marrow.

Keywords: Bone marrow transplantation(骨髓移植), Hematopoesis(造血), HSCs(造血干细胞), Competitive(竞争的), Stem cell engrafment(干细胞植活)

Materials and Reagents

  1. C57BL6 (CD45.2) mice (bred in our laboratory)
  2. C57BL6 (CD45.1) mice (bred in our laboratory)
  3. Ciprofloxacin 200 (200 mg/100 ml Bayer)
  4. Phosphate Buffered Saline (PBS) (without Ca2+ and Mg2+) (Sigma-Aldrich, catalog number: D8537 )
  5. Turk’s solution (Merck KGaA, catalog number: 1.09277.0100 )
  6. Heparin sodium salt (Sigma-Aldrich, catalog number: H3393-100KU )
  7. Ficoll-Paque plus (GE Healthcare, catalog number: 17-1440-03 )
  8. FITC anti-mouse CD45.1 (cloneA20) (Biolegend, catalog number: 110705 )
  9. PE anti-mouse CD115 (clone AFS98) (Biolegend, catalog number: 135506 )
  10. APC anti-mouse B220 (clone RA3-6B) (eBioscience, catalog number: 17-0552-81 )
  11. FITC anti-mouse Gr-1 (clone RB6-8C5) (Biolegend, catalog number: 108405 )
  12. APC anti-mouse Ter119 (clone TER-119) (Biolegend, catalog number: 116211 )
  13. PE anti-mouse CD3 (clone 145- 2C11) (Biolegend, catalog number: 100307 )
  14. APC anti-mouse NK1.1 (clone PK136) (Biolegend, catalog number: 108709 )
  15. Streptavidin PerCp (Biolegend, catalog number: 405213 )
  16. Sodium azide
  17. FACS buffer (see Recipes)
  18. RBC lysis buffer (see Recipes)
  19. Heparin solution (see Recipes)

Equipment

  1. 3 ml syringe (BD Biosciences, catalog number: 305270 )
  2. Forceps and sharp scissors
  3. Needles (21 G x 1.5-Nr.2 0.8 mm x 40 mm) (BD Biosciences, catalog number: 304432 )
  4. 0.5 ml syringes (BD, catalog number: 320933 )
  5. Mouse restrainer
  6. Red light lamp
  7. Tissue culture (TC) dish (6 well plate) (Corning, catalog number: 3516 )
  8. 15 ml conical tubes (BD Biosciences, Falcon®, catalog number: 352196 )
  9. 70 μM nylon strainers (BD Biosciences, Falcon®, catalog number: 352350 )
  10. Inverted microscope
  11. Ice bucket
  12. Hemacytometer (Sigma-Aldrich, catalog number: Z359629-1EA )
  13. Centrifuge
  14. Scalpel knife
  15. Eppendorf tubes (Axygen®, catalog number: MCT-175-C ).
  16. FACS micro-tubes (USA Scientific, catalog number: 1412-1000 )
  17. FACS tubes (BD, catalog number: 352054 )
  18. Mice Irradiation machine (Gamma cell 40 Exactor) (InsideView)
  19. BD LSR II flow cytometer

Software

  1. FACSDiva software
  2. FlowJo software

Procedure

Part I: Bone marrow transplantation
Before starting, it is important to understand that the following protocol is designed to test competitive bone marrow reconstitution of desired transgenic donors. Competition is always with wild-type congenic C57BL6 CD45.1 bone marrow cells. It is crucial to test the competition of appropriate wild-type control cells with the competitor and the comparison is always done between the tested donors and their wild-type controls.


  1. One day prior to transplantation
    Irradiate C57BL6 (CD45.2) host females (5 mice per transplantation group) with 9.5 Gy total body irradiation (TBI), (Gamma cell 40 Exactor machine). Following exposure to TBI, supplement the drinking water with 1:100 dilution of Ciprofloxacin 200. The antibiotic supplemented water should not be exposed to light.
    Note: The mice are 8 weeks old, and are irradiated according to the Gamma cell 40 Exactor machine 9.5 Gy or 950 rad. The time is according to the calibration of the machine.

  2. Day of the transplantation
    1. Sacrifice C57BL6 donor mice (CD45.2) and C57BL6 competitor mice (CD45.1) by CO2 euthanization or cervical dislocation.
    2. Extract the hind limbs using forceps and sharp scissors. Place the bones in PBS on ice. We usually pool bones of 2-3 mice per group, if mice are not to be pooled, separate the individual bones in different wells of a 6 well plate.
    3. It is essential to label the wells in the 6 well plate in order to be able to identify donor (CD45.2) and competitor (CD45.1) bones correctly.
    4. Flush the total bone marrow cells from bone cavity (both limbs of the mouse) with 5 ml ice cold PBS (5 ml in 15 ml tube per mouse) using a 3 ml syringe and 21 G x 1.5 needle. To dissociate the bone marrow cells, resuspend the 5 ml cell solution using the same syringe.
    5. Pass the 5 ml cell suspension trough 70 μM nylon strainer to obtain a uniform single-cell suspension.
    6. Count the cells (5 ml) by diluting the cells 1:5-1:10 with Turk’s solution. Count he cells under the inverted light microscope using the Hemacytometer, count all 9 squares. From one mouse typically one will get 50 million cells from both femurs and tibias.
    7. Counting the cells is a crucial step; a mistake in cell count can give false results. We usually count every sample in duplicates from three independent dilutions with Turk’s solution. (If 1:10 dilution gives a very low count, less than 40 cells, lower the dilution factor. The most accurate count should give between 50-200 cells.).
    8. The cell count in each sample per ml is the number of cells: cells counted x dilution factor x 104.
    9. Competitive BM transplantation is usually achieved by transplanting 1:1 ration of donor versus competitor bone marrow cell. If desired, one can change the transplantation ratio.
    10. Transplanting 1:1 ratio: total of 5 million cells are transplanted to each host; for controls, donor and competitor cells are transplanted separately. (5 host mice per control group).
    11. Prepare cell mixtures in PBS of donor and competitor cells according to the table:

      Experiment group 1: Competitive 1:1 test- group transplantation
      Experiment group 2: Competitive 1:1 control transplantation
      12.5 x 106 cell/ml test donor
      12.5 x 106 cell/ml competitor
      12.5 x 106 cell/ml control donor
      12.5 x 106 cell/ml competitor

    12. Load 0.2 ml into 0.5 ml syringes. It may be prudent to prepare extra syringes for each group. Having one extra can help as tail vein injections can be tricky.
    13. Inject 0.2 ml of the cell mixture into each recipient animal tail vein (5 for each group).
      For the tail vein injection, the mice are heated slightly using an infra-red lamp, restrained and the cells injected into the tail vein of the mice.
    14. Recipient mice should be congenic wild-type C57BL6 mice (CD45.2).
    15. As mentioned, these animals need to be irradiated with 9.5 Gy of irradiation 24 h prior to injection.
    16. Following injection, the recipient mice are left to rest for 4-6 weeks. Water should be changed to normal after a week.

Part II: Blood reconstitution analysis
First analysis of total blood donor reconstitution is evaluated between 4-6 weeks following bone marrow transplantation. We usually start at 6 weeks and monitor reconstitution, once every two weeks up to 20 weeks.


  1. Obtaining total white blood cells for flow cytometry
    1. Heat the mice with a red lamp (be careful not to over-heat the mice).
    2. Place mouse in a strainer.
    3. With a sharp scalpel knife, nick the tail vein and collect up to 200 µl blood into properly labeled eppendorf tube (do not extract more than around 200 µl of blood per mouse).
    4. Make sure to add 30 µl of 10,000 U/ml Heparin solution to each eppendorf tube before collecting blood. This will prevent coagulation of the blood.
    5. Keep doing the same for all the recipient mice of the experiment.
    6. Carefully add 200 µl PBS to every blood sample, if the eppendorf tube is dirty, change it.
    7. Carefully pipette (very slowly) 400 µl ficoll to the bottom of each tube. Two phases should form; ficoll phase at the bottom and blood phase at the top.
    8. Centrifuge the tubes at 4,000 x g for 6 min at room temperature.
    9. Carefully remove the tubes from the centrifuge; the bone marrow mono-nuclear cells will form a fine white ring between the two phases, around 200 µl volume.
    10. Carefully collect the ring of cells with 200 µl pipette tip into a new FACS micro-tube.
    11. Divide each sample to two FACS micro-tubes, if additional cell staining is desired (optional).
    12. Make sure to carefully label the FACS micro-tubes, to properly identify the samples.
    13. Wash the cells once with cold FACS buffer: add 1 ml FACS buffer to each tube.
    14. Centrifuge the cells, 200 x g, 5 min at 4 °C.
    15. Carefully aspirate the supernatant using a vacuum, the cell pellet will most likely not be visible therefore, aspirate the supernatant leaving enough liquid at the bottom, do not aspirate all the liquid!

  2. Labeling cells for flow cytometry
    1. Dilute Biotin anti-mouse CD45.1 antibody in FACS buffer (1:100); calculate volume for the number of samples needed, usually prepare excess volume (for example: For 20 samples, make 25x solution - 500 µl FACS buffer and 5 µl antibody).
    2. Resuspend the cells in 20 µl of Antibody solution prepared; add the antibody directly to the cells. Do not add to tube wall.
    3. Vortex carefully every tube to resuspend the cells.
    4. Incubate on ice for 10 min.
    5. Add 1 ml FACS Buffer to each tube.
    6. Centrifuge the cells, 200 x g, 5 min at 4 °C.
    7. Carefully aspirate the supernatant.
    8. Flow cytometry staining can be done as follows:

      Labeling #1: 
      Myeloid populations labeling (20 µl vol/tube)
      Labeling #2:
      Lymphoid populations labeling (20 µl vol/tube)
      PE anti-mouse CD115 antibody (diluted 1:250)
      FITC anti-mouse Gr-1 antibody (diluted 1:100)
      APC anti-mouse Ter119 antibody (diluted 1:100)
      Streptavidin PerCp
      APC anti-mouse B220 antibody (dilute 1:400)
      PE anti-mouse CD3 antibody (dilute 1:200)
      FITC anti-mouse NK1.1 antibody (dilute 1:100)
      Streptavidin PerCp

    9. Make a stock solution with FACS Buffer for each staining according to the number of samples.
    10. Re-suspend each sample directly with 20 µl antibody solution.
    11. Vortex carefully each tube.
    12. Protect from light.
    13. Incubate on ice for 10 min.
    14. Add 1 ml FACS Buffer to each tube.
    15. Centrifuge the cells, 200 x g, 5 minutes at 4 °C.
    16. Carefully aspirate the supernatant.

  3. Flow cytometry
    1. On the BD LSR II software, select for a 'New Experiment' to set up.
    2. Select for Area, Height, Width for FSC and SSC; select for Log and Area for the colors FITC, PE, APC, PerCP.
    3. Create compensation controls and adjust the gating for unstained blood cells (adjust FSC and SSC voltages as necessary).
    4. Adjust each color of single stains in the voltage panel so that the positive peak is at the 104 mark.
    5. For single stains, use anti-CD45.1 for PerCP, anti-B220 for APC, anti-CD3 for PE and Anti-Gr-1 for FITC.
    6. Record the desired voltages after any adjustments for each of the single stains and then calculate compensation controls.
    7. Events are now ready to be recorded-set up to collect 5,000-10,000 event.

Part III: Long-term bone marrow engraftment analysis

  1. Sacrifice the chimeras 20 weeks post transplantation by CO2 euthanization or cervical dislocation.
  2. Extract the hind limbs using forceps and sharp scissors. Place the bones in PBS on ice. Separate the individual bones in different wells of a 6 well plate.
  3. It is essential to label the wells in the 6 well plates in order to be able to identify bones correctly.
  4. Flush the total bone marrow cells from bone cavity with 5 ml ice cold PBS (15 ml tube per sample) using a 3 ml syringe and 21 G x 1.5 needle. To dissociate the bone marrow cells, resuspend the 5 ml cell solution using the same syringe.
  5. Centrifuge the cells 200 x g, 5 min at room temperature.
  6. Re-suspend the pellet in 1 ml RBC lysis buffer.
  7. Incubate the cells 1.5 min on ice.
  8. Add 10 ml PBS to each tube.
  9. Centrifuge the cells 200 x g, 5 min at room temperature.
  10. Re-suspend the cell pellet in 5 ml PBS.
  11. Pass the 5 ml cell suspension trough 50 μM nylon strainer to obtain a uniform single-cell suspension.
  12. Transfer 1 ml of each sample to FACS tubes.
  13. Make sure to carefully label the FACS tubes, to properly identify the samples.
  14. Centrifuge the cells, 200 x g, 5 min at 4 °C.
  15. Proceed to flow cytometry staining as described previously.

Recipes

  1. FACS buffer
    0.5% fetal calf serum (FCS)
    2 mM EDTA
    0.05% sodium azide
    Dissolved in sterile PBS
    Filter sterilize
  2. RBC lysis buffer
    0.15 M NH4Cl
    1 mM NaHCO3
    0.1 mM EDTA dissolved in sterile double distilled water
    Adjust pH to 7.2-7.4 with 1 M HCl
    Filter sterilize
  3. Heparin solution
    Dissolve the 100 KU Heparin sodium salt in 10 ml sterile double distilled water to obtain 10,000 U/ml

Acknowledgments

The protocol was adapted from the previously published paper: Maryanovich et al. (2012). Our studies are supported in part by the Israel Science Foundation (ISF), USA-Israel Binational Science Foundation (BSF), German-Israel Foundation (GIF), Minerva Stiftung, MDM ICR Research Award, and Milgrom Award. A.G. is the incumbent of the Armour Family Career Development Chair of Cancer Research.

References

  1. Maryanovich, M., Oberkovitz, G., Niv, H., Vorobiyov, L., Zaltsman, Y., Brenner, O., Lapidot, T., Jung, S. and Gross, A. (2012). The ATM-BID pathway regulates quiescence and survival of haematopoietic stem cells. Nat Cell Biol 14(5): 535-541.

简介

竞争性骨髓移植测定测量照射的移植受体小鼠中造血的多谱系重建。 因此,该测定常规用于在体内确定造血干细胞和祖细胞(HSPC)功能。 该方法的原理是将骨髓供体细胞(源自选择的转基因小鼠)与正常竞争剂骨髓一起移植到C57BL6背景上。 为了在移植时区分供体和竞争者细胞,通常竞争性小鼠是同基因的并携带最初称为Ly5.1和CD45.1的差别B细胞抗原。典型的竞争性骨髓移植实验将含有两个移植组, 供体(选择的转基因小鼠及其对照)与正常竞争剂竞争移植,并在血液和骨髓中评价移入效率。

关键字:骨髓移植, 造血, 造血干细胞, 竞争的, 干细胞植活

材料和试剂

  1. C57BL6(CD45.2)小鼠(在我们的实验室培育)
  2. C57BL6(CD45.1)小鼠(在我们的实验室培育)
  3. 环丙沙星200(200mg/100ml Bayer)
  4. 磷酸盐缓冲盐水(PBS)(不含Ca 2+和Mg 2+ 2+)(Sigma-Aldrich,目录号:D8537)
  5. Turk溶液(Merck KGaA,目录号:1.09277.0100)
  6. 肝素钠盐(Sigma-Aldrich,目录号:H3393-100KU)
  7. Ficoll-Paque plus(GE Healthcare,目录号:17-1440-03)
  8. FITC抗小鼠CD45.1(克隆A20)(Biolegend,目录号:110705)
  9. PE抗小鼠CD115(克隆AFS98)(Biolegend,目录号:135506)
  10. APC抗小鼠B220(克隆RA3-6B)(eBioscience,目录号:17-0552-81)
  11. FITC抗小鼠Gr-1(克隆RB6-8C5)(Biolegend,目录号:108405)
  12. APC抗小鼠Ter119(克隆TER-119)(Biolegend,目录号:116211)
  13. PE抗小鼠CD3(克隆145-2C111)(Biolegend,目录号:100307)
  14. APC抗小鼠NK1.1(克隆PK136)(Biolegend,目录号:108709)
  15. 链霉亲和素PerCp(Biolegend,目录号:405213)
  16. 叠氮化钠
  17. FACS缓冲区(参见配方)
  18. RBC裂解缓冲液(参见配方)
  19. 肝素溶液(见配方)

设备

  1. 3ml注射器(BD Biosciences,目录号:305270)
  2. 镊子和锋利的剪刀
  3. 针(21G×1.5-Nr.2.8mm×40mm)(BD Biosciences,目录号:304432)
  4. 0.5ml注射器(BD,目录号:320933)
  5. 鼠标限制器
  6. 红光灯
  7. 组织培养(TC)皿(6孔板)(Corning,目录号:3516)
  8. 15ml锥形管(BD Biosciences,Falcon ,目录号:352196)
  9. 70μM尼龙滤器(BD Biosciences,Falcon ,目录号:352350)
  10. 倒置显微镜
  11. 冰桶
  12. 血细胞计数器(Sigma-Aldrich,目录号:Z359629-1EA)
  13. 离心机
  14. 手术刀
  15. Eppendorf管(Axygen ,目录号:MCT-175-C)。
  16. FACS微管(USA Scientific,目录号:1412-1000)
  17. FACS管(BD,目录号:352054)
  18. 小鼠照射机(Gamma cell 40 Exactor)(InsideView)
  19. BD LSR II流式细胞仪

软件

  1. FACSDiva软件
  2. FlowJo软件

程序

第I部分 :骨髓移植
在开始之前,重要的是要理解以下方案设计用于测试期望的转基因供体的竞争性骨髓重建。竞争总是与野生型同类C57BL6 CD45.1骨髓细胞。用竞争剂测试合适的野生型对照细胞的竞争是至关重要的,并且总是在测试的供体和它们的野生型对照之间进行比较。


  1. 移植前一天
    用9.5Gy全身照射(TBI),(Gamma细胞40外泌机)照射辐射C57BL6(CD45.2)宿主雌性(每个移植组5只小鼠)。在暴露于TBI后,用1:100稀释的环丙沙星200补充饮用水。补充抗生素的水不应暴露于光。
    注意:小鼠是8周龄,并根据Gamma细胞40生物反应器机器9.5Gy或950rad辐照。时间是根据机器的校准。

  2. 移植日期
    1. 通过CO 2安乐死或颈脱位来牺牲C57BL6供体小鼠(CD45.2)和C57BL6竞争小鼠(CD45.1)。
    2. 使用镊子和锋利的剪刀提取后肢。将骨头放在冰上的PBS中。我们通常将每组2-3只小鼠的骨骼混合,如果不合并小鼠,则在6孔板的不同孔中分离单独的骨。
    3. 为了能够正确识别供体(CD45.2)和竞争对手(CD45.1)的骨骼,必须在6孔板中标记孔。
    4. 使用3ml注射器和21G×1.5针,用5ml冰冷PBS(5ml,每只小鼠15ml试管)从骨腔(小鼠的两肢)冲洗总骨髓细胞。为了分离骨髓细胞,使用相同的注射器重悬5ml的细胞溶液
    5. 将5毫升细胞悬浮液通过70微米尼龙过滤器,得到均匀的单细胞悬浮液
    6. 通过用Turk's溶液稀释细胞1:5-1:10计数细胞(5ml)。使用血细胞计数器在倒置光学显微镜下计数细胞,计数所有9个正方形。从一只小鼠,通常将从股骨和胫骨获得50百万个细胞。
    7. 计数细胞是关键的一步;细胞计数的错误可能会产生错误的结果。我们通常使用Turk的解决方案对来自三次独立稀释的重复样本进行计数。 (如果1:10稀释得到非常低的计数,小于40个细胞,降低稀释因子,最准确的计数应该在50-200个细胞之间)。
    8. 每个样品中每毫升的细胞计数是细胞数:细胞计数x稀释因子×10 4 。
    9. 竞争性BM移植通常通过移植1:1比例的供体与竞争性骨髓细胞来实现。如果需要,可以改变移植比例。
    10. 移植1:1比率:将总共500万个细胞移植到每个宿主;对于对照,分别移植供体和竞争细胞。 (每个对照组5个宿主小鼠)。
    11. 根据下表制备供体和竞争细胞的PBS中的细胞混合物:

      实验组1:竞争性1:1测试组移植
      实验组2:竞争性1:1控制移植
      12.5×10 6个细胞/ml受试供体
      12.5×10 6个细胞/ml竞争剂
      12.5×10 6个细胞/ml对照供体
      12.5×10 6个细胞/ml竞争剂

    12. 加载0.2毫升到0.5毫升注射器。 为每个组准备额外的注射器可能是谨慎的。 有一个额外的可以帮助作为尾静脉注射可以是棘手的。
    13. 将0.2ml细胞混合物注入每个受体动物尾静脉(每组5只) 对于尾静脉注射,使用红外灯将小鼠轻微加热,约束并将细胞注射到小鼠的尾静脉中。
    14. 受体小鼠应该是同类野生型C57BL6小鼠(CD45.2)
    15. 如上所述,这些动物需要在注射前24小时用9.5Gy的照射进行照射
    16. 注射后,使受体小鼠静止4-6周。 一周后水应改为正常。

第二部分:血液重建分析
在骨髓移植后4-6周之间评估总供体重建的第一分析。 我们通常在6周开始,监测重建,每两周一次,最多20周。


  1. 获得用于流式细胞术的总白细胞
    1. 用红灯加热小鼠(小心不要过热小鼠)。
    2. 将鼠标放在过滤器中。
    3. 用锋利的手术刀切开尾静脉,收集200μl血液到正确标记的eppendorf管中(每只小鼠不要提取超过约200μl的血液)。
    4. 确保在收集血液之前向每个eppendorf管中加入30μl的10,000 U/ml肝素溶液。 这将防止血液凝结。
    5. 对于实验的所有受体小鼠,保持相同。
    6. 小心地为每个血液样品添加200μlPBS,如果eppendorf管脏了,改变它
    7. 小心移液(非常缓慢)400微升ficoll到每个管的底部。 两相应形成; 底部有聚蔗糖相,顶部有血液相
    8. 在室温下以4,000×g离心管6分钟。
    9. 小心地从离心机中取出试管; 骨髓单核细胞将在两个相之间形成细小的白色环,大约200μl体积。
    10. 小心收集细胞的环用200微升移液器吸头到新的FACS微管
    11. 如果需要额外的细胞染色(可选),将每个样品分成两个FACS微管。
    12. 确保仔细标记FACS微管,以正确识别样品
    13. 用冷的FACS缓冲液洗涤细胞一次:向每个管中加入1ml FACS缓冲液
    14. 在4℃下将细胞离心200分钟,5分钟。
    15. 使用真空小心地吸出上清液,细胞沉淀将很可能是不可见的,因此,吸出上清液留下底部足够的液体,不吸取所有的液体!

  2. 标记细胞用于流式细胞术
    1. 在FACS缓冲液(1:100)中稀释生物素抗小鼠CD45.1抗体; 计算所需样品数量的体积,通常制备过量(例如:对于20个样品,制备25x溶液 - 500μlFACS缓冲液和5μl抗体)。
    2. 重悬细胞在20微升的抗体溶液制备; 将抗体直接加入细胞。 不要添加到管壁。
    3. 小心涡旋每个管以重悬细胞。
    4. 在冰上孵育10分钟。
    5. 向每个管中加入1ml FACS缓冲液
    6. 在4℃下将细胞离心200分钟,5分钟。
    7. 小心吸出上清液。
    8. 流式细胞术染色可以如下进行:

      标签#1:
      骨髓群体标记(20μl体积/管)
      标签#2:
      淋巴细胞群标记(20μl体积/管)
      PE抗小鼠CD115抗体(1:250稀释)
      FITC抗小鼠Gr-1抗体(1:100稀释)
      APC抗小鼠Ter119抗体(稀释1:100)
      链霉亲和素PerCp
      APC抗小鼠B220抗体(稀释1:400)
      PE抗小鼠CD3抗体(稀释1:200)
      FITC抗小鼠NK1.1抗体(稀释1:100)
      链霉亲和素PerCp

    9. 根据样品数量,为每次染色用FACS缓冲液制备储备溶液
    10. 用20μl抗体溶液直接重悬每个样品
    11. 小心涡旋每个管。
    12. 避光保护。
    13. 在冰上孵育10分钟。
    14. 向每个管中加入1ml FACS缓冲液
    15. 在4℃下离心细胞,200×g/g,5分钟。
    16. 小心吸出上清液。

  3. 流式细胞术
    1. 在BD LSR II软件上,选择"新实验"进行设置。
    2. 选择面积,高度,宽度的FSC和SSC; 为颜色FITC,PE,APC,PerCP选择日志和区域。
    3. 创建补偿控制和调整未染色血细胞的门控(必要时调整FSC和SSC电压)。
    4. 调整电压面板中单个污渍的每种颜色,使正峰位于10 4 标记处。
    5. 对于单染色,对于PerCP使用抗CD45.1,对于APC使用抗B220,对于PE使用抗CD3,以及对FITC使用抗Gr-1。
    6. 在对每个单个污渍进行任何调整后记录所需的电压,然后计算补偿控制
    7. 活动现在已准备好记录 - 设置为收集5,000-10,000个活动。

第三部分:长期骨髓移植分析

  1. 通过CO 2安乐死或颈脱位移植后20周牺牲嵌合体。
  2. 使用镊子和锋利的剪刀提取后肢。 将骨头放在冰上的PBS中。 分离在6孔板的不同井中的单个骨。
  3. 为了能够正确识别骨骼,必须在6孔板中标记孔。
  4. 使用3ml注射器和21G×1.5针,用5ml冰冷PBS(每个样品15ml管)冲洗来自骨腔的总骨髓细胞。 为了分离骨髓细胞,使用相同的注射器重悬5ml的细胞溶液
  5. 在室温下将细胞离心200分钟,5分钟。
  6. 将沉淀重悬在1ml RBC裂解缓冲液中。
  7. 在冰上孵育细胞1.5分钟。
  8. 每管加入10ml PBS。
  9. 在室温下将细胞离心200分钟,5分钟。
  10. 将细胞沉淀重悬在5ml PBS中
  11. 将5毫升细胞悬浮液通过50微米的尼龙过滤器,得到均匀的单细胞悬浮液
  12. 转移1毫升的每个样品到FACS管
  13. 确保仔细地标记FACS管,以正确识别样品
  14. 在4℃下将细胞离心200分钟,5分钟。
  15. 继续如前所述的流式细胞术染色

食谱

  1. FACS缓冲区
    0.5%胎牛血清(FCS)
    2mM EDTA 0.05%叠氮化钠 溶于无菌PBS中
    过滤灭菌
  2. RBC裂解缓冲液
    0.15 M NH 4 Cl
    1mM NaHCO 3/v/v 0.1mM EDTA溶于无菌双蒸水中 用1M HCl将pH调节至7.2-7.4 过滤灭菌
  3. 肝素溶液
    将100KU肝素钠盐溶于10ml无菌双蒸水中,得到10,000U/ml

致谢

该协议改编自以前发表的文章:Maryanovich等人(2012)。 我们的研究部分由以色列科学基金会(ISF),美国 - 以色列二元科学基金会(BSF),德以色列基金会(GIF),Minerva基金会,MDM ICR研究奖和Milgrom奖支持。 A.G.是Armour家庭职业发展癌症研究主席的任职者。  

参考文献

  1. Maryanovich,M.,Oberkovitz,G.,Niv,H.,Vorobiyov,L.,Zaltsman,Y.,Brenner,O.,Lapidot,T.,Jung,S.and Gross,A。 ATM-BID途径调节造血干细胞的静止和存活。 Nat Cell Biol 14(5):535-541。
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引用:Maryanovich, M. and Gross, A. (2014). Competitive Bone-marrow Transplantations. Bio-protocol 4(11): e1145. DOI: 10.21769/BioProtoc.1145.
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