In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells
原代小鼠骨髓细胞诱导生成体外破骨细胞试验   

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Nature Cell Biology
Oct 2017

 

Abstract

Osteoclasts are a group of bone-absorbing cells to degenerate bone matrix and play pivotal roles in bone growth and homeostasis. The unbalanced induction of osteoclast differentiation (osteoclastogenesis) in pathological conditions, such as osteoporosis, arthritis and skeleton metastasis of cancer, causes great pain, bone fracture, hypercalcemia or even death to patients. In vitro osteoclastogenesis analysis is useful to better understand osteoclast formation in physiological and pathological conditions. Here we summarized an easy-to-follow osteoclastogenesis protocol, which is suitable to evaluate the effect of different factors (cytokines, small molecular chemicals and conditioned medium from cell culture) on osteoclast differentiation using primary murine bone marrow cells.

Keywords: Osteoclastogenesis (破骨细胞生成), Osteoclast (破骨细胞), Primary bone marrow (原代骨髓), TRAP staining (TRAP染色), Cell culture (细胞培养)

Background

The skeleton is maintained by successive and well-controlled absorbance and formation of bone mass during lifetime. In the bone cavity, each of these two activities is carried out by a specialized cell type: the bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts and osteoclasts are derived from bone-resident mesenchymal cells and hematopoietic lineage progenitor cells, respectively. The differentiation of hematopoietic myeloid progenitor cells into mature osteoclasts are majorly controlled by receptor activator of nuclear factor-κB ligand (RANKL, encoded by TNFSF11) and macrophage colony-stimulating factor (M-CSF, encoded by CSF1) derived from osteoblasts and its progenitor cells (Suda et al., 1999). Unlike other cells, osteoclasts differentiate through fusion of a certain number of progenitor cells (Boyle et al., 2003). Thus, a key histological feature of mature osteoclasts is their multiple nuclei. After maturation, osteoclasts are capable of bone resorption by producing an acidified microenvironment to dissolve bone mass mainly composed of calcium phosphate, along with proteases to degrade extracellular matrix (Boyle et al., 2003). The dissolved bone matrix releases sequestered growth factors utilized by osteoblasts to expand their population (Kassem and Bianco, 2015). This cross-talk between osteoblasts and osteoclasts ensures coordinate bone-forming and -degenerating activity, which is dysregulated in a plethora of diseases, including osteoporosis, arthritis and bone metastasis of cancers (Rodan and Martin, 2000; Raisz, 2005; Gupta and Massague, 2006). Based on previous literature (Lu et al., 2009; Wang et al., 2014; Zhuang et al., 2017), here we describe a step-by-step protocol for an in vitro osteoclastogenesis assay using primary murine bone marrow cells that allows studying the effect of a broad range of factors/conditions (such as cytokines and conditioned medium) on osteoclast differentiation.

Materials and Reagents

  1. Pipet tips (Autoclaved, any brand)
  2. 29 gauge syringe (BD, catalog number: 328421 )
  3. 40 µm nylon mesh cell strainer (Corning, catalog number: 352340 )
  4. Minisart® NML syringe filter, 0.2 µm (Sartorius, catalog number: 17597-K )
  5. 14 mm round coverslips (any brand suitable for cell culture)
  6. 24 well plate and 100 mm Petri dish (Thermo Fisher Scientific, NuncTM, catalog numbers: 142475 and 172931 )
  7. 15 ml conical tubes (Corning, catalog number: 352196 )
  8. Glass slides (OMANO, catalog number: OMSK-50PL )
  9. Cell culture Petri dishes and multi-well plate (Thermo Fisher Science, catalog numbers: 174888 , 150350 , 142485 )
  10. 1.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )
  11. Mouse aged between 4-7 weeks (any eligible provider, mouse strain/genetic background should be consistent with other assays)
  12. α-MEM (Thermo Fisher Scientific, catalog number: A1049001 )
  13. Fetal bovine serum (Thermo Fisher Scientific, catalog number: 10099141 )
  14. Recombinant murine M-CSF (PeproTech, catalog number: 315-02 )
  15. Recombinant murine RANKL (PeproTech, catalog number: 315-11 )
  16. Red blood cell lysing buffer (BD, catalog number: 555899 )
  17. Albumin, bovine serum, fraction V (Merck, catalog number: 12659 )
  18. Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma-Aldrich, catalog number: 387A )
  19. Acetone (Fisher Scientific, catalog number: A18-1 )
  20. 37% formaldehyde (Fisher Scientific, catalog number: BP531-500 )
  21. Neutral Balsam (Sangon Biotech, catalog number: E675007 )
  22. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
  23. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  24. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
  25. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  26. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H9892 )
  27. Culture medium (see Recipes)
  28. Phosphate buffered saline (see Recipes)
  29. Murine rRANKL and rM-CSF solution (see Recipes)

Equipment

  1. Pipette (Eppendorf, sterilize surface by 70% ethanol)
  2. Hemacytometer (OMANO, catalog number: OMSK-HEMA )
  3. Dissecting tweezers and scissors (Autoclaved)
    Scissors (Fisher Scientific, catalog number: 08-940 )
    Tweezers (Fisher Scientific, catalog number: 12-460-612)
    Manufacturer: Integra LifeSciences, Integra® Miltex®, catalog number: MH18782 .
    Tweezers (Fisher Scientific, catalog number: 12-460-611)
    Manufacturer: Integra LifeSciences, Integra® Miltex®, catalog number: MH18780 .
  4. Cell culture incubator (any brand)
  5. Centrifuge (Thermo Fisher Scientific, model: Hearus Labofuge 400 R )
  6. Balance
  7. Inverted microscope (Nikon Instruments, model: Eclipse Ti-S )
  8. Water bath (any brand which can reach 37 °C)

Procedure

Note: For the animal experiment included in the protocol, please refer to specific regulations in your institute. In our study, all animal studies were conducted according to the guidelines for the care and use of laboratory animals and were approved by the Institutional Biomedical Research Ethics Committee.

  1. Day 0: Extraction of bone marrow cells
    Note: The volumes of medium or buffer in the procedures are for one mouse. Adjust the volume if handling multiple mice.
    1. Sacrifice a mouse aged between 4-7 weeks by cervical dislocation and quickly sterilize the body surface with 70% ethanol.
    2. Immediately dissect out the hinder limbs of the mouse by cutting at the ankle and the upper end of the thigh with scissors. Remove skin and muscle to reveal bones. Dislocate the femur and tibia by cutting at the knee with scissors. Rinse bones twice with pre-cooled PBS to remove excessive muscle tissues (Figure 1A).
    3. Remove both ends of each piece of bone (Figure 1B) and flush the bone marrow cells out with a 29 gauge syringe. Use 0.5 ml α-MEM per bone and temporarily collect cell solution in a 6 cm Petri dish (Figure 1C).
      Note: If bone marrow cells do not flow out easily, stir within the bone cavity with the syringe needle briefly to mobilize cells.
    4. Pipet the resultant cell solution gently and filter it through a 40 µm mesh cell strainer (Figure 1D) into a 15 ml conical tube. This step is to remove unwanted cell clusters and bone matrix. Collect cells via centrifugation at 400 x g for 5 min at 4 °C and discard supernatant.
    5. Resuspend cell pellets in 200 µl α-MEM and add 2 ml diluted red blood cell lysing buffer. Gently mix the solution by inverting the tube a couple of times and then leave at room temperature for 5-10 min until the solution becomes transparent red (Figure 1E).
      Note: Prepare the red blood cell lysing buffer just before use. Red blood cell lysing buffer is supplied as 10x stock. Dilute it with deionized water to the working concentration.
    6. Collect cells by centrifugation at 400 x g for 5 min at 4 °C. Cell pellets should be almost white at this stage. If not, repeat Steps A5 and A6.
    7. Discard supernatant and resuspend cells in 10 ml culture medium and culture in a 10 cm Petri dish in a 37 °C, 5% CO2 cell culture incubator overnight.


      Figure 1. Overview of osteoclastogenesis assay. A. Dissected femur and tibia; B. Removal of bone ends; C. Flushing of bone marrow cells; D. Filtering through a cell strainer; E. Primary bone marrow cells before (left), during (middle) and after (right) red blood cell lysis; F. Representative image of mature osteoclasts. Scale bar = 50 µm.

  2. Day 1: Osteoclastogenesis
    1. Collect suspended myeloid precursor cells in a 15 ml conical tube and centrifuge at 400 x g for 5 min at 4 °C.
    2. Resuspend cell pellets in 1 ml culture medium and count cell number by hemacytometer.
      Note: Usually 1-2 x 107 cells can be obtained with one mouse at this step.
    3. Place sterilized coverslips into the central 8 wells of 24 well plates.
      Note: Usually four replicates are required for each setting.
    4. Dilute cell solution with culture medium or culture medium/conditioned medium mixture to the concentration of 1.6-2.0 x 106/ml. Aliquot 2 ml cell solution for each setting (four replicates). Add M-CSF (final concentration, 25 ng/ml) and RANKL (final concentration, 50 ng/ml) stock to their final concentration.
      Note: The final concentration of rRANKL may vary and please refer to Notes section for guidance.
      If specific cytokines or chemicals are of interest, please also add them into the cell aliquots. Gently but thoroughly pipet the cell solution and transfer 500 µl mixtures to each of the four replicates.

  3. Day 4: Refresh medium with the same medium recipe as Day 1

  4. Day 7: TRAP staining for mature osteoclasts
    1. Conduct staining procedures of coverslips following the manufacturer’s protocol (Sigma). Here is the Sigma protocol with our modification:
      Note: The whole fixation and staining procedures can be conducted in the same multi-well plate of osteoclast differentiation.
      1. Prepare Fixative Solution by combining 25 ml Citrate solution (provided by the staining kit), 65 ml acetone and 8 ml 37% formaldehyde. Place Fixative Solution in glass bottles and cap tightly. Fixative solution is stable up to 2 months at 2-8 °C and discard if evaporation is observed.
      2. Pre-warm sufficient deionized water (at least 10 ml for each well) to 37 °C for the whole assay, including washes before and after staining.
      3. Bring Fixative Solution to room temperature (18-26 °C). Aspirate culture medium and rinse once with PBS. Fix cells on coverslips by adding 500 µl Fixative Solution/well for 30 sec. Rinse thoroughly in deionized water: Do not allow slides to dry.
      4. To a 1.5 ml Eppendorf tube add 0.5 ml Fast Garnet GBC Base Solution and 0.5 ml Sodium Nitrite Solution. Mix by gentle inversion for 30 sec. Let it stand for 2 min.
      5. Prepare staining solution in the “Beaker B” column (45 ml deionized water pre-warmed to 37 °C, 1.0 ml diazotized Fast Garnet GBC solution from Step D1d, 0.5 ml Naphthol AS-BI Phosphate solution, 2.0 ml Acetate Solution and 1.0 ml Tartrate Solution).
        Note: All solutions except deionized water are provided by the staining kit.
      6. Add at least 500 µl staining solution to each well. Place multi-well plates in a 37 °C incubator for 1 h and protect plates from light.
      7. Aspirate staining solution and rinse several times with deionized water. Carefully take cover slips out and air dry them.
    2. Mount of air-dried coverslips
      1. Take a clean glass slide and place one drop of 20 µl neutral balsam onto the slide.
      2. Carefully hold coverslips on the edge, facing the surface with cells toward glass slide, and slowly mount to avoid air bubbles.
      3. Count results within one week after mounting. Mature osteoclasts are TRAP-positive cells with ≥ 3 nuclei and diameter ≥ 50 µm (Figure 1F).
      Note: We usually count TRAP-positive multi-nuclei cells of a whole coverslip to collect data. It is not suggested to collect data by taking random snapshots since it is very difficult to pre-determine how many snapshots are sufficient to estimate the number of osteoclasts.

Notes

  1. The administration of antibiotics may cause unsuccessful induction of osteoclasts and should be avoided. Please use sterile equipment to handle mouse tissue and cells. The conical tubes, syringes, Petri dishes, multi-well plates and cell strainers mentioned in the Materials and Reagents section are all sterilized. Other tools, such as tips, dissecting tweezers, scissors, and coverslips can be autoclaved. Equipment which cannot be autoclaved, such as pipettes, can be sterilized by 70% ethanol spraying. After the equipment has been properly handled, the risk of contamination should be quite low.
  2. The quality of FBS may strongly affect the differentiation of osteoclasts. Also, the rRANKL final concentration can vary from 0 to 100 ng/ml due to the different quality of FBS, and 50 ng/ml should be an appropriate point to start with. Users need to adjust the concentration of rRANKL since too much rRANKL can mask the modulation caused by the specific reagents which users are interested in. Users can test a series of concentrations ranging from 0 to 100 ng/ml in pilot trials. The appropriate concentration should 1) allow users to observe mature osteoclasts with the right morphology and 2) not saturate osteoclastogenesis, which means increased rRANKL administration can still induce more osteoclasts.

Recipes

  1. Culture medium
    α-MEM with 20% heat-inactivated FBS
    Note: FBS can be heat-inactivated by incubation at 56 °C for 30 min.
  2. Phosphate buffered saline
    1 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Diluted in 800 ml deionized water and adjust to pH 7.4 with HCl
    Add deionized water to the final volume of 1 L and autoclave the solution
  3. Murine rRANKL and rM-CSF solution
    1. Dissolve 0.5 g BSA in 10 ml sterilized PBS and filter through a 0.2 µm filter
    2. Reconstitute lyophilized protein powder in PBS containing 0.5% BSA solution to the final concentration of 0.1 mg/ml and gently agitate to ensure it is fully dissolved
    3. Aliquot cytokine solution and store at -80 °C

Acknowledgments

This work was funded by the National Natural Science Foundation of China (81430070, 81661148048, 81725017), the Chinese Academy of Sciences (QYZDB-SSW-SMC013, XDA12050101) and the Ministry of Science and Technology of China (2017YFA0103502). This work is adapted from previous literature (Lu et al., 2009; Wang et al., 2014). Authors declare no conflict of interest or competing interests.

References

  1. Boyle, W. J., Simonet, W. S. and Lacey, D. L. (2003). Osteoclast differentiation and activation. Nature 423(6937): 337-342.
  2. Gupta, G. P. and Massague, J. (2006). Cancer metastasis: building a framework. Cell 127(4): 679-695.
  3. Kassem, M. and Bianco, P. (2015). Skeletal stem cells in space and time. Cell 160(1-2): 17-19.
  4. Lu, X., Wang, Q., Hu, G., Van Poznak, C., Fleisher, M., Reiss, M., Massague, J. and Kang, Y. (2009). ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis. Genes Dev 23(16): 1882-1894.
  5. Raisz, L. G. (2005). Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest 115(12): 3318-3325.
  6. Rodan, G. A. and Martin, T. J. (2000). Therapeutic approaches to bone diseases. Science 289(5484): 1508-1514.
  7. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T. and Martin, T. J. (1999). Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20(3): 345-357.
  8. Wang, Y., Lei, R., Zhuang, X., Zhang, N., Pan, H., Li, G., Hu, J., Pan, X., Tao, Q., Fu, D., Xiao, J., Chin, Y. E., Kang, Y., Yang, Q. and Hu, G. (2014). DLC1-dependent parathyroid hormone-like hormone inhibition suppresses breast cancer bone metastasis. J Clin Invest 124(4): 1646-1659.
  9. Zhuang, X., Zhang, H., Li, X., Li, X., Cong, M., Peng, F., Yu, J., Zhang, X., Yang, Q., and Hu, G. (2017). Differential effects on lung and bone metastasis of breast cancer by Wnt signalling inhibitor DKK1. Nat Cell Biol 19(10): 1274-1285.

简介

破骨细胞是一组骨吸收细胞,用于退化骨基质并在骨生长和体内平衡中发挥关键作用。 在诸如骨质疏松症,关节炎和癌症骨骼转移等病理状态下破骨细胞分化(破骨细胞生成)的不平衡诱导会导致严重疼痛,骨折,高钙血症或甚至导致患者死亡。 体外破骨细胞生成分析对于更好地理解生理和病理条件下的破骨细胞形成是有用的。 在这里,我们总结了一种易于遵循的破骨细胞生成方案,其适用于评估不同因素(细胞因子,小分子化学物质和来自细胞培养物的条件培养基)对使用原代鼠骨髓细胞的破骨细胞分化的影响。

【背景】骨骼在生命周期内通过连续且良好控制的吸光度和骨量形成来维持。在骨腔中,这两种活性中的每一种都是通过特定的细胞类型进行的:骨形成性成骨细胞和骨降解性破骨细胞。成骨细胞和破骨细胞分别来自骨驻留间充质细胞和造血谱系祖细胞。造血系统骨髓祖细胞分化为成熟破骨细胞主要受核因子-κB配体受体激活因子(RANKL,由TNFSF11编码)和巨噬细胞集落刺激因子(M-CSF,由< )来自成骨细胞及其祖细胞(Suda et al。 ,1999)。与其他细胞不同,破骨细胞通过融合一定数量的祖细胞而分化(Boyle等人,2003)。因此,成熟破骨细胞的关键组织学特征是它们的多个核。成熟后,破骨细胞能够通过产生酸化的微环境来溶解主要由磷酸钙组成的骨质以及蛋白酶以降解细胞外基质(Boyle等人,2003),从而能够骨吸收。溶解的骨基质释放成骨细胞使用的隔离生长因子以扩大其人群(Kassem和Bianco,2015)。成骨细胞和破骨细胞之间的这种相互作用确保了协调的骨形成和 - 退化活性,其在包括骨质疏松症,关节炎和癌症骨转移的过多疾病中失调(Rodan和Martin,2000; Raisz,2005; Gupta和Massague ,2006)。根据之前的文献(Lu等人,2009; Wang等人,2014; Zhuang等人,2017),在此我们描述使用原代鼠骨髓细胞进行体外破骨细胞发生试验的逐步方案,其允许研究广泛范围的因子/条件(例如细胞因子和条件培养基)对破骨细胞的影响分化。

关键字:破骨细胞生成, 破骨细胞, 原代骨髓, TRAP染色, 细胞培养

材料和试剂

  1. 移液技巧(高压灭菌,任何品牌)
  2. 29号注射器(BD,目录号:328421)

  3. 40μm尼龙网格细胞过滤器(Corning,目录号:352340)
  4. Minisart NML注射器过滤器,0.2μm(Sartorius,目录号:17597-K)
  5. 14毫米圆盖玻片(适用于细胞培养的任何品牌)
  6. 24孔板和100mm培养皿(Thermo Fisher Scientific,Nunc TM,目录号:142475和172931)。

  7. 15毫升锥形管(Corning,目录号:352196)
  8. 玻璃幻灯片(OMANO,目录号:OMSK-50PL)
  9. 细胞培养培养皿和多孔板(Thermo Fisher Science,目录号:174888,150350,142485)
  10. 1.5 ml Eppendorf管(Fisher Scientific,目录号:05-408-129)

  11. 小鼠4-7周龄(任何合格的供应商,小鼠品系/遗传背景应与其他测定一致)
  12. α-MEM(Thermo Fisher Scientific,目录号:A1049001)
  13. 胎牛血清(Thermo Fisher Scientific,目录号:10099141)
  14. 重组鼠M-CSF(PeproTech,目录号:315-02)
  15. 重组小鼠RANKL(PeproTech,目录号:315-11)
  16. 红细胞裂解缓冲液(BD,目录号:555899)
  17. 白蛋白,牛血清,V级(Merck,目录号:12659)
  18. 酸性磷酸酶,白细胞(TRAP)试剂盒(Sigma-Aldrich,目录号:387A)
  19. 丙酮(Fisher Scientific,目录号:A18-1)
  20. 37%甲醛(Fisher Scientific,目录号:BP531-500)
  21. 中性香脂(Sangon Biotech,产品目录号:E675007)
  22. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358)
  23. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  24. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S5136)
  25. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  26. 盐酸(HCl)(Sigma-Aldrich,目录号:H9892)
  27. 培养基(见食谱)
  28. 磷酸盐缓冲盐水(见食谱)
  29. 鼠rRANKL和rM-CSF溶液(见食谱)

设备

  1. 移液器(Eppendorf,70%乙醇消毒表面)
  2. 血细胞计数器(OMANO,目录号:OMSK-HEMA)
  3. 解剖镊子和剪刀(高压灭菌)
    剪刀(Fisher Scientific,产品目录号:08-940)
    镊子(Fisher Scientific,目录号:12-460-612)
    制造商:Integra LifeSciences,Integra ® Miltex ® ,产品目录号:MH18782。
    镊子(Fisher Scientific,目录号:12-460-611)
    制造商:Integra LifeSciences,Integra ® Miltex ® ,产品目录号:MH18780。
  4. 细胞培养孵化器(任何品牌)
  5. 离心机(Thermo Fisher Scientific,型号:Hearus Labofuge 400 R)
  6. 平衡
  7. 倒置显微镜(尼康仪器,型号:Eclipse Ti-S)
  8. 水浴(任何品牌可以达到37°C)

程序

注意:对于协议中包含的动物实验,请参考您所在研究所的具体规定。在我们的研究中,所有的动物研究都是根据实验动物护理和使用指南进行的,并获得了机构生物医学研究伦理委员会的批准。

  1. 第0天:提取骨髓细胞
    注意:程序中的介质或缓冲区的容量适用于一个鼠标。如果处理多个鼠标,请调整音量。
    1. 牺牲4-7周龄的小鼠颈椎脱臼,并迅速用70%乙醇消毒身体表面。
    2. 立即用剪刀在脚踝和大腿的上端切开鼠标的四肢。去除皮肤和肌肉以显示骨骼。用剪刀在膝盖处切断使股骨和胫骨脱位。
      用预先冷却的PBS冲洗骨骼两次以去除过多的肌肉组织(图1A)。
    3. 去除每块骨头的两端(图1B),并用29号注射器冲洗骨髓细胞。每个骨使用0.5ml的α-MEM,并在6cm培养皿中临时收集细胞溶液(图1C)。
      注意:如果骨髓细胞不容易流出,用注射器针头短暂地在骨腔内搅动以调动细胞。
    4. 轻轻吸取得到的细胞溶液,并将其通过40μm网孔细胞过滤器(图1D)过滤到15ml锥形管中。这一步是去除不需要的细胞簇和骨基质。通过在4℃下以400×g离心5分钟收集细胞并丢弃上清液。
    5. 在200μl的α-MEM中重悬细胞沉淀并加入2ml稀释的红细胞裂解缓冲液。通过翻转试管轻轻混合溶液几次,然后在室温下放置5-10分钟,直到溶液变成透明红色(图1E)。
      注意:使用前准备好红细胞裂解缓冲液。红细胞裂解缓冲液以10x储备液供应。用去离子水稀释至工作浓度。
    6. 通过在4℃下以400×g离心5分钟收集细胞。在这个阶段,细胞团应该几乎是白色的。如果不是,请重复步骤A5和A6。
    7. 弃去上清液并将细胞重悬于10ml培养基中,并在37℃,5%CO 2细胞培养箱中于10cm培养皿中培养过夜。


      图1.破骨细胞生成测定概述A.解剖的股骨和胫骨; B.去除骨头; C.骨髓细胞的冲洗; D.通过细胞过滤器过滤; E.在(左),(中)和(右)红细胞溶解期间的原代骨髓细胞; F.成熟破骨细胞的代表性图像。比例尺= 50微米。

  2. 第1天:破骨细胞生成
    1. 将悬浮的骨髓前体细胞收集在15ml锥形管中,并在4℃下以400gxg离心5分钟。
    2. 在1ml培养基中重悬细胞团并通过血细胞计数器计数细胞数量。
      注意:在此步骤中,通常只需一只鼠标即可获得1-2 x 10元的电池。 />
    3. 将消毒后的盖玻片放入24孔板的中心8个孔中。
      注意:每个设置通常需要重复4次。
    4. 用培养基或培养基/条件培养基混合物稀释细胞溶液至浓度为1.6-2.0×10 6 / ml / ml。等分每个设置2ml细胞溶液(四次重复)。将M-CSF(终浓度25ng / ml)和RANKL(终浓度50ng / ml)原液添加至其最终浓度。
      注:rRANKL的最终浓度可能会有所不同,请参阅说明部分以获取指导。
      如果特定细胞因子或化学物质感兴趣,请将它们添加到细胞等分试样中。轻轻但彻底地吸取细胞溶液,并将500μl混合物转移到四次重复中的每一次。

  3. 第4天:使用与第1天相同的中等配方刷新媒体。

  4. 第7天:成熟破骨细胞的TRAP染色
    1. 遵循制造商的协议(Sigma)进行盖玻片的染色步骤。这里是我们修改后的Sigma协议:
      注意:整个固定和染色过程可以在同一个破骨细胞分化的多孔板中进行。
      1. 通过混合25ml柠檬酸盐溶液(由染色试剂盒提供),65ml丙酮和8ml 37%甲醛来制备固定溶液。将固色剂溶液置于玻璃瓶中并盖紧。固定溶液在2-8°C时稳定2个月,如果观察到蒸发,则可丢弃。
      2. 预热足够的去离子水(每孔至少10ml)至整个测定的37℃,包括染色前后的洗涤。
      3. 将固定溶液带到室温(18-26°C)。吸出培养基并用PBS冲洗一次。通过添加500微升固定液/井固定细胞在盖玻片上30秒。
        在去离子水中彻底冲洗:不要让玻片干燥。
      4. 向1.5ml Eppendorf管中加入0.5ml快速石榴石GBC碱溶液和0.5ml亚硝酸钠溶液。轻轻倒转混合30秒。让它站立2分钟。
      5. 在“Beaker B”柱(预热至37℃的45ml去离子水,来自步骤D1d的1.0ml重氮化快速石榴石GBC溶液,0.5ml萘酚AS-BI磷酸盐溶液,2.0ml乙酸盐溶液和1.0ml酒石酸盐溶液)。
        注:除去离子水以外的所有溶液均由染色试剂盒提供。
      6. 每孔加入至少500μl染色溶液。将多孔板置于37°C培养箱中1小时,并保护板免受光照。
      7. 吸去染色液并用去离子水冲洗数次。小心取下盖子,然后风干。
    2. 空气干燥的盖玻片的装载。
      1. 取一张干净的载玻片,并将一滴20μl中性香脂放在载玻片上。
      2. 小心地抓住边缘上的盖玻片,使细胞朝向载玻片朝向表面,并缓慢安装以避免气泡。
      3. 安装后一周内计算结果。成熟的破骨细胞是≥3个核且直径≥50μm的TRAP阳性细胞(图1F)。
      注:我们通常计数整个盖玻片TRAP阳性多核细胞收集数据。不建议通过随机快照收集数据,因为预先确定有多少快照足以估计破骨细胞的数量是非常困难的。

笔记

  1. 施用抗生素可能导致破骨细胞诱导失败,应避免使用。请使用无菌设备来处理鼠标组织和细胞。材料和试剂部分中提到的锥形管,注射器,培养皿,多孔板和细胞过滤器都是经过消毒的。其他工具,如提示,解剖镊子,剪刀和盖玻片可以高压灭菌。不能被高压灭菌的设备,如移液器,可以通过70%的乙醇喷雾灭菌。设备正确处理后,污染风险应该很低。
  2. FBS的质量可能强烈影响破骨细胞的分化。而且,由于FBS的质量不同,rRANKL终浓度可以在0到100ng / ml之间变化,并且50ng / ml应该是适合的开始点。用户需要调整rRANKL的浓度,因为过多的rRANKL可以掩盖由用户感兴趣的特定试剂引起的调节。用户可以在中试中测试0到100ng / ml的一系列浓度。适当的浓度应该1)允许使用者观察具有正确形态的成熟破骨细胞和2)不使饱和破骨细胞形成,这意味着增加的rRANKL施用仍然可以诱导更多的破骨细胞。

食谱

  1. 培养基
    含20%热灭活FBS的α-MEM
    注意:通过在56℃孵育30分钟,可以将FBS热灭活。
  2. 磷酸盐缓冲盐水
    1克NaCl
    0.2克KCl
    1.44克Na 2 HPO 4 4 0.24克KH 2 PO 4 4 用800ml去离子水稀释并用HCl调节至pH7.4 加入去离子水至1L的最终体积并高压灭菌该溶液。
  3. 鼠rRANKL和rM-CSF溶液
    1. 将0.5 g BSA溶解在10 ml无菌PBS中并通过0.2μm过滤器过滤。
    2. 在含有0.5%BSA溶液的PBS中重建冻干蛋白粉至终浓度为0.1mg / ml,轻轻搅动以确保其完全溶解。
    3. 分装细胞因子溶液并储存在-80°C

致谢

本研究由国家自然科学基金(81430070,81661148048,81725017),中国科学院(QYZDB-SSW-SMC013,XDA12050101)和中国科学技术部(2017YFA0103502)资助。这项工作改编自以前的文献(Lu等人,2009; Wang等人,2014)。作者声明不存在利益冲突或利益冲突。

参考

  1. Boyle,W.J.,Simonet,W.S。和Lacey,D.L。(2003)。 破骨细胞分化与激活 Nature 423(6937) :337-342。
  2. Gupta,G.P。和Massague,J。(2006)。 癌症转移:构建框架 Cell 127( 4):679-695。
  3. Kassem,M.和Bianco,P。(2015)。 空间和时间的骨骼干细胞 细胞 160 (1-2):17-19。
  4. Lu,X.,Wang,Q.,Hu,G.,Van Poznak,C.,Fleisher,M.,Reiss,M.,Massague,J。和Kang,Y。(2009)。 ADAMTS1和MMP1通过蛋白水解方式使EGF样配体在骨溶解信号级联反应中发生骨转移。 Genes Dev 23(16):1882-1894。
  5. Raisz,L.G。(2005)。 骨质疏松症的发病机制:概念,冲突和前景。 J Clin Invest 115(12):3318-3325。
  6. Rodan,G.A。和Martin,T.J。(2000)。 治疗骨疾病的方法 Science 289(5484 ):1508-1514。
  7. Suda,T.,Takahashi,N.,Udagawa,N.,Jimi,E.,Gillespie,M.T。和Martin,T.J。(1999)。 肿瘤坏死因子受体和配体家族的新成员调节破骨细胞分化和功能< / a> Endocr Rev 20(3):345-357。
  8. Wang,Y.,Lei,R.,Zhuang,X.,Zhang,N.,Pan,H.,Li,G.,Hu,J.,Pan,X.,Tao,Q.,Fu,D., Xiao,J.,Chin,YE,Kang,Y.,Yang,Q.和Hu,G。(2014)。 DLC1依赖性甲状旁腺激素样激素抑制抑制乳腺癌骨转移 J Clin Invest 124(4):1646-1659。
  9. 壮,X,张,H,李,X,李,X.,丛,M.,彭,楼,余,J.,张,X.,杨,Q.和胡,G。 (2017年)。 Wnt信号抑制剂DKK1对乳腺癌肺和骨转移的不同作用 < Nat Cell Biol 19(10):1274-1285。
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引用:Zhuang, X. and Hu, G. (2018). In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells. Bio-protocol 8(11): e2875. DOI: 10.21769/BioProtoc.2875.
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Benjamin Rodriguez-Garay
CIATEJ
Perra de Hiram
6/17/2018 10:59:27 AM Reply