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Osteoblast Sorting and Intracellular Staining of CXCL12
成骨细胞分选和CXCL12的细胞内染色   

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Cancer Research
Mar 2016

Abstract

Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping. The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are expressed by osteoblasts and bone marrow stroma cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006) and for supporting leukemia initiating cell activity (Pitt et al., 2015). Here we describe the procedure of intracellular staining of CXCL12.

Keywords: Bone marrow niche (骨髓微环境), HSC (HSC), Osteoblast (成骨细胞), CXCL12 (CXCL12), Intracellular staining (细胞内染色 )

Background

The bone marrow niche is a highly organized microenvironment with stroma cells that engage in direct cell-cell interaction with hematopoietic stem cells (HSC) that regulate HSC quiescence, differentiation, and mobilization (Anthony and Link, 2014; Mendelson and Frenette, 2014; Morrison and Scadden, 2014). Multiple cell types in HSC niche may contribute to niche functional support in distinct but maybe overlapping ways. These cells include but are not limited to osteoblasts, osteoclasts, CXCL12-abundant reticular (CAR) cells, Nestin+ stroma cells, leptin receptor+ (LepR+) stroma cells, endothelial cells, macrophages, megakaryocytes, neuronal, and the non-myelinating Schwann cells. Most HSCs are found in the trabecular region of bone marrow, suggesting an important HSC supporting role of the endosteum as well as factors made by osteoblasts and other cells in the endosteum (Kiel et al., 2005; Lo Celso et al., 2009). The majority of long-term HSCs are located in close vicinity of the sinusoid in close contact with LepR+ and CXCL12high niche cells, indicating a perivascular niche composed by endothelial or perivascular cells (Kiel et al., 2005; Sugiyama et al., 2006; Acar et al., 2015). In addition to the perivascular niche associated with sinusoid, mesenchymal cells that surround arterioles in the bone marrow are also important for the maintenance of quiescent HSCs (Kunisaki et al., 2013). Osteoblasts are specialized endosteum-lining cells that are terminally differentiated products of mesenchymal stem cells. Characterization of murine primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone and retrospective functional analysis (Bakker and Klein-Nulend, 2011). However, culture-based analysis has been complicated by the heterogeneity of the tissue. Immunophenotyping of murine osteoblasts based on defined CD markers is a rapid and prospective approach of phenotypic analysis of osteoblasts in various disease processes. Isolated osteoblasts through flow-based sorting are especially suitable for downstream applications such as gene expression analysis.

The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are highly expressed by CAR cells but also by osteoblasts and endothelial cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006). CXCL12 in the vascular niche has also been shown play a critical role in supporting leukemia-initiating cell (LIC) activity (Pitt et al., 2015). Using a mouse T-ALL model, we reported that leukemia development was accompanied by the drastic suppression of the endosteum-lining osteoblast population. We further showed that aberrant Notch activation negatively regulates the expression of CXCL12 and osteoblastic progenitor differentiation. Here we describe the procedure of sorting mouse bone marrow osteoblastic cells and the procedure of staining intracellular CXCL12 (Wang et al., 2016).

Materials and Reagents

  1. Gauze sponges (Fisher Scientific, FisherbrandTM, catalog number: 22-415-468 )
  2. 21 G needles
  3. 3 ml syringes
  4. 70 μm strainer (Fisher Scientific, FisherbrandTM, catalog number: 22-363-548 )
  5. 1.5 ml microcentrifuge tube (NEST Biotechnology, catalog number: 615601 ), 15 ml tubes (BioExpress, GeneMateTM, catalog number: C-3394-1 ), and 50 ml conical tubes (BioExpress, GeneMateTM, catalog number: C-3394-4 )
  6. Other antibodies:
    APC-anti-CD31 (Thermo Fisher Scientific, eBioScienceTM, catalog number: 17-0311-82 )
    PE-anti-CD51 (BD, BD PharmingenTM, catalog number: 551187 )
    PE-Cy7-anti-CD45 (BD, BD PharmingenTM, catalog number: 552848 )
    Biotin-Sca1 (BD, BD PharmingenTM, catalog number: 553334 )
    Streptavidin APC-Cy7 (BD, BD PharmingenTM, catalog number: 554063 )
  7. CXCL12 detection antibodies: H/M CXCL12/SDF-1 Fluorescein (FITC) MAb (Clone 79018) (R&D Systems, catalog number: IC350F )
  8. 70% (v/v) ethanol
  9. Hank’s Balanced Salt Solution (HBSS; 1x) (GE Healthcare, HycloneTM, catalog number: SH30588.02 )
  10. BSA (Sigma-Aldrich, catalog number: A7906 )
  11. Type I collagenase (Worthington, Lakewood, NJ)
  12. DMEM (ATCC®, catalog number: 30-2002TM )
  13. Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD, BD Cytofix/Cytoperm™ Plus, catalog number: 554715 )
  14. HBSS staining buffer (see Recipes)

Equipment

  1. Pipettes
  2. Scissors
  3. Tweezers
  4. Curved forceps
  5. Tabletop centrifuge (SORVALL Legend RT)
  6. Hemocytometer
  7. FACSAria I

Procedure

  1. Preparing single cell suspension from long bones and osteoblast sorting
    1. Euthanize the mouse by CO2 asphyxiation and confirmed death by cervical dislocation.
    2. Sterilize the body with 70% (v/v) ethanol.
    3. Make incisions around the connection between hind limbs and trunk using scissors and tweezers. The whole skin is then removed from the hind limbs by pulling toward the cutting site of the claw (Figure 1A). Carefully remove all the muscles from the long bones (femur and tibia) using dissecting scissors and curved forceps. Excise the long bone and transfer onto sterile gauze.
    4. Clean soft tissue as thoroughly as possible around the bone (Figure 1B).


      Figure 1. Preparation of mouse long bones. A. Exposure of hind legs. B. Cleaned long bones (tibia and femur) after removing attached muscles.

    5. Cut off the epiphyses.
    6. Fill a 21-gauge needle and 3 ml syringe with 1 ml cold HBSS supplemented with 0.5% BSA and use a syringe to flush out bone marrow (BM) three times (Figure 2A).
    7. Cut the bone into small pieces of approximately 1-2 mm2 using scissors (Figure 2B).


      Figure 2. Preparation of long bone fragments. A. Cleaned long bones after flushing out bone marrow; B. Prepared bone fragments.

    8. Digest bone fragments obtained from 2 sets of tibias and femurs per mouse in 2 ml HBSS buffer supplemented with collagenase I (3 mg/ml in DMEM) in a 50 ml conical tube for 1 h by shaking (110 rpm) at 37 °C.
    9. Add 3 volume of DMEM (6 ml) supplemented with 10% FBS to terminate collagenase I activity.
    10. Strain the cell solution through a 70 μm strainer in a 50 ml conical tube.
    11. Spin down at 250 x g for 5 min at 4 °C.
    12. Remove supernatant
    13. Wash the cells with 2 ml of HBSS buffer and spin down at 250-400 x g for 5 min at 4 °C.
    14. Discard supernatant and resuspend the cells in 1 ml HBSS buffer.
    15. Determine cell number. Count cells using a hemocytometer.
    16. Spin down at 250-400 x g for 5 min at 4 °C.
    17. The cells are ready for preparing for osteoblast sorting and CXCL12 staining.
    18. Prepare antibody cocktails in HBSS buffer using the following antibodies: FITC- lineage markers (T cells: CD4, CD8; B cells: B220; erythrocytes: Ter119; granulocytes and monocytes: CD11b, and Gr-1), APC-CD31 (endothelial cell), PE-CD51, PE-Cy7-Sca1. Use 100 µl of HBSS buffer containing 1 µl of each antibody for 1 x 106 cells.
    19. Resuspend cells from Step A17 in antibody mixture on ice for 20 min.
    20. Wash cells twice each with 200-500 µl cold HBSS buffer, spinning down at 250-400 x g for 5 min at 4 °C in between washes.
    21. Sort cells with FACSAria I. Osteoblasts are sorted by gating on lin- (CD4, CD8, B220, Ter119, CD11b, Gr-1) CD31-CD51+Sca1- population (Figures 3A and 3B).


      Figure 3. Sorting of osteoblasts. A. Gating strategy of lin- (CD4, CD8, B220, Ter119, CD11b, Gr-1) CD31- population; B. Gated osteoblasts (lin- CD31-CD51+Sca1-).

  2. CXCL12/SDF-1 Intracellular Staining
  1. Resuspend cells from Step A17 from procedure A in HBSS buffer in a 15 ml Falcon tube. Add biotin-Sca1 antibody and leave cells on ice for 20 min. Use 100 µl of HBSS buffer containing 0.5 µl of antibody for 1 x 106 cells.
  2. Wash cells twice each with 200-500 µl HBSS buffer.
  3. Spin down at 250 x g for 5 min at 4 °C. Resuspend cells in HBSS buffer and add APC-CD31, PE-CD51, PE-Cy7-CD45, and Streptavidin APC-Cy7 antibodies, and then incubate on ice for 20 min.
  4. Wash twice each with 1 ml HBSS buffer. Spin down cells at 250 x g for 5 min.
  5. Fix and permeabilize cells by resuspending cells in 250 µl of Fixation/Permeabilization solution (included in the BD Cytofix/CytopermTM Plus) for 15-20 min at 4 °C.
    Note: Cell aggregation can be avoided by gently vortexing prior to the addition of the Fixation/Permeabilization solution.
  6. Wash cells twice each with 1 ml of 1x Perm/WashTM solution (included in the BD Cytofix/CytopermTM Plus), pellet cells by centrifugation at 250 x g for 5 min at 4 °C, and remove supernatant.
    Note: Perm/WashTM solution is required in washing steps to maintain cells in a permeabilized state.
  7. Stain intracellular CXCL12 by adding 10 μl of FITC-CXCL12 in 90 μl 1x Perm/WashTM solution and vortex gently.
  8. Incubate cells for 30 min at room temperature in a dark room.
  9. Wash cells twice each with 1 ml 1x Perm/WashTM Buffer. Spin down cells at 250 x g for 5 min at 4 °C.
  10. Resuspend cells in 200 μl HBSS buffer for flow cytometric analysis (see Figure 4 below).
    Note: For a negative control, a separate set of cells should be stained with an isotype control antibody.

Data analysis

From wild type (WT) mice long bones, osteoblasts are readily isolated from the bone marrow stroma after brief digestion with collagenase I (Figure 4A). In comparison, osteoblasts are decreased in the marrow stroma from mice developing T-ALL driven by activated Notch1 (Figure 4B). CXCL12 expression by intracellular staining and flow analysis shows 52% decrease in leukemia mice osteoblasts compared to normal controls (Figure 4C).


Figure 4. Sorting and CXCL12 staining of bone marrow osteoblasts. Bone marrow stroma cells were prepared from WT or leukemia-developing mice. Staining of marrow osteoblast (OB) (lin-CD31- CD51+Sca-1-) (A and B) by gating on the lineage-CD31-popualtion. Intracellular expression of CXCL12 was displayed by mean fluorescence intensity (MFI) using isotype control or anti-CXCL12 antibody (C).

Recipes

  1. HBSS staining buffer
    Hank’s Balanced Salt Solution (HBSS; 1x) supplemented with 0.5% BSA

Acknowledgments

This work was supported by grants from American Cancer Society LIB-125064 (L. Zhou) and NIH HL103827 (L. Zhou). No potential conflicts of interest were disclosed.

References

  1. Anthony, B. A. and Link, D. C. (2014). Regulation of hematopoietic stem cells by bone marrow stromal cells. Trends Immunol 35(1): 32-37.
  2. Acar, M., Kocherlakota, K. S., Murphy, M. M., Peyer, J. G., Oguro, H., Inra, C. N., Jaiyeola, C., Zhao, Z., Luby-Phelps, K. and Morrison, S. J. (2015). Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526(7571): 126-130.
  3. Bakker, A. D. and Klein-Nulend, J. (2011). Osteoblast isolation from murine calvaria and long bones. Methods Mol Biol 816: 19-29.
  4. Kiel, M. J., Yilmaz, O. H., Iwashita, T., Yilmaz, O. H., Terhorst, C. and Morrison, S. J. (2005). SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121(7): 1109-1121.
  5. Kunisaki, Y., Bruns, I., Scheiermann, C., Ahmed, J., Pinho, S., Zhang, D., Mizoguchi, T., Wei, Q., Lucas, D., Ito, K., Mar, J. C., Bergman, A. and Frenette, P. S. (2013). Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502(7473): 637-643.
  6. Lo Celso, C., Fleming, H. E., Wu, J. W., Zhao, C. X., Miake-Lye, S., Fujisaki, J., Cote, D., Rowe, D. W., Lin, C. P. and Scadden, D. T. (2009). Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457(7225): 92-96.
  7. Mendelson, A. and Frenette, P. S. (2014). Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med 20(8): 833-846.
  8. Morrison, S. J. and Scadden, D. T. (2014). The bone marrow niche for haematopoietic stem cells. Nature 505(7483): 327-334.
  9. Pitt, L. A., Tikhonova, A. N., Hu, H., Trimarchi, T., King, B., Gong, Y., Sanchez-Martin, M., Tsirigos, A., Littman, D. R., Ferrando, A. A., Morrison, S. J., Fooksman, D. R., Aifantis, I. and Schwab, S. R. (2015). CXCL12-producing vascular endothelial niches control acute T cell leukemia maintenance. Cancer Cell 27(6): 755-768.
  10. Sugiyama, T., Kohara, H., Noda, M. and Nagasawa, T. (2006). Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 25(6): 977-988.
  11. Wang, W., Zimmerman, G., Huang, X., Yu, S., Myers, J., Wang, Y., Moreton, S., Nthale, J., Awadallah, A., Beck, R., Xin, W., Wald, D., Huang, A. Y. and Zhou, L. (2016). Aberrant notch signaling in the bone marrow microenvironment of acute lymphoid leukemia suppresses osteoblast-mediated support of hematopoietic niche function. Cancer Res 76(6): 1641-1652.

简介

成骨细胞是通过分泌因子和细胞粘附分子在调节造血干细胞中发挥重要作用的骨髓内皮细胞生态位细胞。 通过培养胶原酶处理的骨骼的生长已经实现了原代成骨细胞的表征。 长骨成骨细胞的免疫分型和基于流动的分析为表征成骨细胞提供了简化和快速的方法。 我们描述了基于细胞表面免疫分型的分离小鼠骨髓成骨细胞的修改过程。 趋化因子CXCL12(也称为基质衍生因子SDF-1)与其受体CXCR4一起由成骨细胞和骨髓基质细胞表达。 CXCL12-CXCR4轴对于造血干细胞滞留于其生态位(Sugiyama等,2006)和支持白血病启动细胞活性(Pitt等,2015年)。 这里我们描述CXCL12细胞内染色的过程。

【背景】骨髓龛是一种高度组织化的微环境,基质细胞参与与调节HSC静止,分化和动员的造血干细胞(HSC)的直接细胞 - 细胞相互作用(Anthony and Link,2014; Mendelson and Frenette,2014; Morrison和斯卡登,2014年)。 HSC细胞生态位中的多种细胞类型可能以不同的但可能重叠的方式贡献于小生境功能支持。这些细胞包括但不限于成骨细胞,破骨细胞,富含CXCL12的网状(CAR)细胞,Nestin +基质细胞,瘦素受体+(LepR + )基质细胞,内皮细胞,巨噬细胞,巨核细胞,神经元和非髓鞘施旺细胞。大多数造血干细胞存在于骨髓的骨小梁区,这表明骨内膜的重要HSC支持作用以及成骨细胞和其他细胞在内骨中产生的因子(Kiel等人,2005; Lo Celso 等人,2009)。大多数长期HSCs位于正弦曲线附近并与LepR +和CXCL12高生态细胞紧密接触,表明由内皮或血管周围组成的血管周细胞生态位细胞(Kiel等,2005; Sugiyama等,2006; Acar等,,2015)。除了与窦状隙相关的血管周细胞小生境外,围绕骨髓中小动脉的间充质细胞对于维持静止的HSC也是重要的(Kunisaki et al。,2013)。成骨细胞是特化的骨内膜细胞,其是间充质干细胞的终末分化产物。小鼠原代成骨细胞的表征通过培养胶原酶处理过的骨的生长和回顾性功能分析来实现(Bakker和Klein-Nulend,2011)。然而,基于文化的分析由于组织的异质性而变得复杂。基于定义的CD标记的小鼠成骨细胞的免疫表型分析是各种疾病过程中成骨细胞的表型分析的快速和前瞻性方法。通过基于流动分选的成骨细胞特别适合下游应用,例如基因表达分析。

趋化因子CXCL12(也称为基质衍生因子SDF-1)与其受体CXCR4一起由CAR细胞高效表达,但也由成骨细胞和内皮细胞高表达。 CXCL12-CXCR4轴对于造血干细胞滞留于其生态位是重要的(Sugiyama等,2006)。血管生态位中的CXCL12也被证明在支持白血病起始细胞(LIC)活性中发挥重要作用(Pitt等人,2015年)。我们使用小鼠T-ALL模型报道了白血病发展伴随着内皮膜成骨细胞群体的剧烈抑制。我们进一步表明异常Notch激活负调控CXCL12和成骨祖细胞分化的表达。在这里,我们描述了分选小鼠骨髓成骨细胞的过程和染色细胞内CXCL12的过程(Wang等人,2016年)。

关键字:骨髓微环境, HSC, 成骨细胞, CXCL12, 细胞内染色

材料和试剂

  1. 纱布海绵(Fisher Scientific,Fisherbrand TM,目录号:22-415-468)
  2. 21 G针头
  3. 3毫升注射器
  4. 70微米过滤器(Fisher Scientific,Fisherbrand TM,目录号:22-363-548)
  5. 1.5ml微量离心管(NEST Biotechnology,目录号:615601),15ml试管(BioExpress,GeneMate TM,目录号:C-3394-1)和50ml锥形管(BioExpress,GeneMate TM ,产品目录号:C-3394-4)
  6. 其他抗体:
    APC抗CD31(Thermo Fisher Scientific,eBioScience TM,目录号:17-0311-82)
    PE-anti-CD51(BD,BD Pharmingen TM,目录号:551187)
    PE-Cy7-抗-CD45(BD,BD Pharmingen TM,目录号:552848)
    生物素-Sca1(BD,BD Pharmingen TM,目录号:553334)
    链霉亲和素APC-Cy7(BD,BD Pharmingen TM,目录号:554063)
  7. CXCL12检测抗体:H / M CXCL12 / SDF-1荧光素(FITC)MAb(克隆79018)(R& D Systems,目录号:IC350F)
  8. 70%(v / v)乙醇
  9. Hank平衡盐溶液(HBSS; 1x)(GE Healthcare,Hyclone TM,目录号:SH30588.02)
  10. BSA(Sigma-Aldrich,目录号:A7906)
  11. I型胶原酶(Worthington,Lakewood,NJ)
  12. DMEM(ATCC ,产品目录号:30-2002 TM)
  13. 具有BD GolgiStopTM(BD,BD Cytofix / Cytoperm™Plus,产品目录号:554715)的固定/透化溶液套件
  14. HBSS染色缓冲液(见食谱)

设备

  1. 移液器
  2. 剪刀
  3. 镊子
  4. 弧形钳
  5. 台式离心机(SORVALL Legend RT)
  6. 血细胞计数器
  7. FACSAria I

程序

  1. 从长骨准备单细胞悬液和成骨细胞分选
    1. 通过CO 2窒息安乐死小鼠并证实颈椎脱臼致死。
    2. 用70%(v / v)乙醇消毒身体。
    3. 用剪刀和镊子在后肢和躯干之间连接切口。通过向爪的切割位置拉动,将整个皮肤从后肢移除(图1A)。使用解剖剪刀和弯钳,小心地去除长骨(股骨和胫骨)上的所有肌肉。切开长骨并转移到无菌纱布上。

    4. 尽可能彻底地清洁软组织(图1B)。


      图1.制备小鼠长骨A.暴露后腿。 B.清除附着的肌肉后清理长骨(胫骨和股骨)。

    5. 切掉骨骺。
    6. 用1 ml冷HBSS(补充有0.5%BSA)填充21号针头和3 ml注射器,并使用注射器冲洗骨髓(BM)三次(图2A)。

    7. 用剪刀将骨切成约1-2mm2的小块(图2B)。


      图2.长骨碎片的制备A.清洗骨髓后清理长骨; B.准备好的骨碎片。

    8. 在37℃下通过振荡(110rpm)在50ml锥形管中补充胶原酶I(3mg / ml,在DMEM中)的2ml HBSS缓冲液中从每组2只胫骨和股骨获得消化骨碎片1小时。
    9. 加入3体积的补充有10%FBS的DMEM(6ml)以终止胶原酶I活性。
    10. 将细胞溶液通过50毫升锥形管中的70μm过滤器过滤。
    11. 在250℃旋转5分钟,在4℃下进行5分钟。
    12. 去除上清液
    13. 用2ml HBSS缓冲液洗涤细胞,并在4℃下以250-400gxg的速度旋转5分钟。
    14. 弃去上清液并将细胞重悬于1ml HBSS缓冲液中。
    15. 确定细胞数量。用血细胞计数器计数细胞。
    16. 在4℃下,以250-400×g 速度旋转5分钟。
    17. 细胞已准备好用于成骨细胞分选和CXCL12染色。
    18. 使用以下抗体制备HBSS缓冲液中的抗体混合物:FITC谱系标记(T细胞:CD4,CD8; B细胞:B220;红细胞:Ter119;粒细胞和单核细胞:CD11b和Gr-1),APC-CD31(内皮细胞),PE-CD51,PE-Cy7-Sca1。使用100μl含有1μl每种抗体的HBSS缓冲液用于1×10 6个细胞。
    19. 将抗体混合物中步骤A17的细胞重悬于冰上20分钟。
    20. 用200-500μl冷HBSS缓冲液洗涤细胞各两次,在4℃在洗涤之间在250-400×g下旋转5分钟。
    21. 用FACSAria I对细胞进行分选。通过门控lin-sup(CD4,CD8,B220,Ter119,CD11b,Gr-1)CD31 - CD51 + Sca1 - 人口(图3A和3B)。


      图3.对成骨细胞的分选A. lin-sup的选择策略(CD4,CD8,B220,Ter119,CD11b,Gr-1)CD31 - / - 人口; B.门控成骨细胞(lin-CD31-CD51 + Sca1-)。

  2. CXCL12 / SDF-1细胞内染色
  1. 在15ml Falcon管中重悬步骤A的步骤A17的HBSS缓冲液中的细胞。加入生物素-Sca1抗体并在冰上放置细胞20分钟。使用100μl含有0.5μl抗体的HBSS缓冲液用于1×10 6个细胞。
  2. 用200-500μlHBSS缓冲液洗涤细胞两次。
  3. 在250℃旋转5分钟,在4℃下进行5分钟。在HBSS缓冲液中重悬细胞并加入APC-CD31,PE-CD51,PE-Cy7-CD45和链霉亲和素APC-Cy7抗体,然后在冰上孵育20分钟。
  4. 每次用1 ml HBSS缓冲液洗两次。将细胞以250×g离心5分钟。
  5. 通过将细胞重悬于250μlFixation /透化溶液(包含在BD Cytofix / Cytoperm TM Plus中)在4℃下15-20分钟来固定和透化细胞。
    注意:在添加Fixation / Permeabilization解决方案之前,可以通过轻轻涡旋来避免细胞聚集。
  6. 用1ml的1x Perm / Wash TM溶液(包含在BD Cytofix / Cytoperm TM Plus中)洗涤细胞两次,通过在250μgxg 在4℃下5分钟,并除去上清液。
    注意:在洗涤步骤中需要Perm / Wash TM TM溶液以保持细胞处于透化状态。 />
  7. 通过在90μl1x Perm / Wash TM溶液中加入10μlFITC-CXCL12染色细胞内CXCL12并轻轻涡旋。

  8. 在室温下孵育细胞30分钟
  9. 用1ml 1x Perm / Wash TM缓冲液洗涤细胞两次。
    在250°C下将细胞旋转5分钟
  10. 将细胞重悬于200μlHBSS缓冲液中进行流式细胞分析(见下图4)。
    注意:对于阴性对照,一组单独的细胞应该用同种型对照抗体染色。

数据分析

从野生型(WT)小鼠长骨中,在用胶原酶I短暂消化后,成骨细胞容易从骨髓基质分离(图4A)。相比之下,在由活化的Notch1驱动的T-ALL小鼠的骨髓基质中成骨细胞减少(图4B)。通过细胞内染色和流动分析的CXCL12表达显示与正常对照相比,白血病小鼠成骨细胞减少52%(图4C)。


图4.骨髓成骨细胞的分选和CXCL12染色从WT或白血病发展小鼠制备骨髓基质细胞。骨髓成骨细胞(OB)(lin-CD31-CD51 + Sca-1-t)的染色(A和B)通过选择谱系 - CD31 - 弹出窗口。通过使用同种型对照或抗CXCL12抗体(C)的平均荧光强度(MFI)显示CXCL12的细胞内表达。

食谱

  1. HBSS染色缓冲液
    Hank平衡盐溶液(HBSS; 1x)补充0.5%BSA

致谢

这项工作得到了美国癌症协会LIB-125064(L. Zhou)和NIH HL103827(L. Zhou)的资助。没有披露任何潜在的利益冲突。

参考

  1. Anthony,B.A。和Link,D.C。(2014)。 骨髓基质细胞对造血干细胞的调节 Trends Immunol 35(1):32-37。
  2. Acar,M.,Kocherlakota,K. S.,Murphy,M. M.,Peyer,J. G.,Oguro,H.,Inra,C. N.,Jaiyeola,C.,Zhao,Z.,Luby-Phelps,K.和Morrison,S.J。(2015)。 骨髓深度显像显示非分裂干细胞主要为周围窦。 自然 526(7571):126-130。
  3. Bakker,A.D。和Klein-Nulend,J。(2011)。 从小鼠颅盖骨和长骨中分离成骨细胞方法Mol Biol 816:19-29。
  4. Kiel,M.J.,Yilmaz,O.H.,Iwashita,T.,Yilmaz,O.H.,Terhorst,C。和Morrison,S.J。(2005)。 SLAM家族受体区分造血干细胞和祖细胞并显示干细胞的内皮龛。 Cell 121(7):1109-1121。
  5. Kunisaki,Y.,Bruns,I.,Scheiermann,C.,Ahmed,J.,Pinho,S.,Zhang,D.,Mizoguchi,T.,Wei,Q.,Lucas,D.,Ito,K., Mar,JC,Bergman,A。和Frenette,PS(2013)。 小动脉生态位维持造血干细胞静止。 Nature 502 (7473):637-643。
  6. Lo Celso,C.,Fleming,H.E.,Wu,J.W.,Zhao,C.X.,Miake-Lye,S.,Fujisaki,J.,Cote,D.,Rowe,D.W.,Lin,C.P.and Scadden,D.T。(2009)。 活体动物跟踪个体造血干/祖细胞在他们的生态位。 >自然 457(7225):92-96。
  7. Mendelson,A.和Frenette,P. S.(2014年)。 体内平衡和再生过程中的造血干细胞小生境维持 Nat Med 20(8):833-846。
  8. Morrison,S.J。和Scadden,D.T。(2014)。 造血干细胞的骨髓龛 Nature 505(7483):327-334。
  9. Pitt,LA,Tikhonova,AN,Hu,H.,Trimarchi,T.,King,B.,Gong,Y.,Sanchez-Martin,M.,Tsirigos,A.,Littman,DR,Ferrando,AA,Morrison, SJ,Fooksman,DR,Aifantis,I.和Schwab,SR(2015)。 产生CXCL12的血管内皮龛可控制急性T细胞白血病的维持。 Cell 27(6):755-768。
  10. Sugiyama,T.,Kohara,H.,Noda,M.和Nagasawa,T。(2006)。 CXCL12-CXCR4趋化因子信号通路在骨髓基质细胞生态位中维持造血干细胞库。 / a> Immunity 25(6):977-988。
  11. Wang,W.,Zimmerman,G.,Huang,X.,Yu,S.,Myers,J.,Wang,Y.,Moreton,S.,Nthale,J.,Awadallah,A.,Beck,R., Xin,W.,Wald,D.,Huang,AY和Zhou,L。(2016)。 急性淋巴细胞白血病骨髓微环境中的异常信号抑制成骨细胞介导的造血龛功能支持。 癌症研究 76(6):1641-1652。
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引用:Wang, W., Majhail, G., Lui, C. and Zhou, L. (2018). Osteoblast Sorting and Intracellular Staining of CXCL12. Bio-protocol 8(10): e2858. DOI: 10.21769/BioProtoc.2858.
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