Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts

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The FASEB Journal
May 2015



Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of osteoclast precursors (Ross et al., 2006; Teitelbaum et al., 2000) (2), and bone resorption pit assay (3).

Keywords: Osteoclast function (破骨细胞功能), Bone resorption (骨吸收), Pit assay (坑试验)

Materials and Reagents

  1. Parafilm
  2. Razor blade
  3. Tissue culture tubes (standard 15 or 50 ml conical TC tubes)
  4. 5 ml syringe with 25 G needle
  5. Cell strainer (70 μm) (Corning, catalog number: 352350 )
  6. Cell culture 96 well plates
  7. Tissue culture petri dish
  8. Whatman filter paper (Sigma-Aldrich, catalog number: WHA10347509 )
  9. 6- to 10-week-old mice
  10. Phosphate buffered saline (PBS), sterilized, pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023 )
  11. Dulbecco's modified Eagle medium, high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  12. Penicillin/streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140-122 )
  13. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  14. MEMα, nucleosides (Thermo Fisher Scientific, catalog number: 12571-063 )
  15. Macrophage-stimulating factor (M-CSF) (PeproTech, catalog number: 315-02 ) and receptor activated nuclear factor k-B ligand (RANKL) (PeproTech, catalog number: 315-11 )
  16. Bovine femurs (from meat market)
  17. Ethanol
  18. Glutaraldehyde (Sigma-Aldrich, catalog number: G5882 )
  19. Toluidine blue (Sigma-Aldrich, catalog number: T3260-5G )
  20. MilliQ water
  21. Acid phosphatase, leukocyte (TRAP) Kit (Sigma-Aldrich, catalog number: 387A-1KT )
  22. Sodium borate 10-hydrate (Fisher Scientific, catalog number: 02-003-999 )
  23. Trypan-blue (Thermo Fisher Scientific, catalog number: T6146-5G )
  24. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434-500G )
  25. Trizma® hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941-100G )
  26. Red blood cell (RBC) lysis buffer (Sigma-Aldrich, catalog number: R7757 ) (see Recipes)
  27. Osteoclast precursor culture media (see Recipes)
  28. Osteoclast differentiation media (see Recipes)
  29. 1% toluidine blue (see Recipes)
  30. Glutaraldehyde solution (see Recipes)


  1. Forceps scissors
  2. Hacksaw
  3. Scalpel
  4. Water ultrasonicator
  5. Isomet low speed saw (BUEHLER, catalog number: 111280 ) with wafering blade
  6. Light microscope


  1. Preparation of bony slices
    Note: If you have pre-made bone slices, please skip to step B.
    1. The diaphysis of bovine femurs is cut transversely into 2-3 cm cylinders with a hacksaw as shown in Figure 1A.
    2. Cut the cylinder into three segments and lift the marrow out as shown in Figure 1B. Any adherent muscle, marrow or periosteum is cleaned off with a scalpel.

      Figure 1. Illustration of transverse and segmental cuts of bovine femur. A. An image of bovine femur depicting diaphyseal portions that are transversely cut into 2-3 cm cylinders; B. Bony cylinders referred in (A) are further cut into three segments as indicated (arrows).

    3. The segments are then sonicated in warm water with a little detergent several times until the bone is clean. Repeat two to three times until the bone is clean. In the original experiment, segments were sonicated for 2 min at 50-60 Hz.
    4. Bony segments are given a final clean of any adherent tissue with a scalpel, wrapped in parafilm and stored under frozen condition (-20 °C). Segments from the same cylinder are given the same label.
    5. To make 4.4 x 4.4 x 0.2 mm bone slices, bony segment is transversely cut into 200 µm thick block with the Isomet low speed saw with the wafering blade. These 200 µm slices are used to produce the final 4.4 x 4.4 x 0.2 mm bone slices.
    6. The stack of 200 µm slices was then clamped and a strip 4.4 mm wide cut was made. 
      The stack of bone strips is then clamped at 90 degrees to the clamp and the final bone slices are produced by cutting at 4.4 mm intervals. These cuttings of the bony segments finally led to the produce of 4.4 x 4.4 x 0.2 mm bone slices.
    7. Store bone slices in 70% ethanol until use.

  2. Preparation of bone marrow-derived osteoclast precursors
    1. Dissect femur and tibia from 6- to 10-week-old mice under sterile condition and place them in cold PBS. Remove all soft tissues, such as muscle, fat, etc. with forceps, scissors and razor blade.
    2. Wash femurs and tibia with sterile PBS twice and cut both their ends.
    3. Flush marrow cells into tissue culture tubes (standard 15 or 50 ml conical TC tubes) from both ends with 5 ml syringe (25 G needle) and DMEM (supplemented with P/S and 1% FBS). About 40 ml DMEM (supplemented with P/S and 1% FBS) is needed to flush marrow cells.
    4. Pelletize marrow cells by centrifuging 637 x g for 5 min at room temperature and discard the supernatant; Re-suspend cells into 3 ml of red blood cell (RBC) lysis buffer at room temperature for 3-4 min to destroy red blood cells.
    5. Add 7 ml αMEM media (with 10% FBS) to cells to neutralize the effect of RBC buffer; Pass the cells through a 70 µm cell strainer into a 50 ml conical tube.
    6. Pelletize cells by centrifuging 637 x g for 5 min at room temperature and discard the supernatant; Wash cells once with 10 ml sterilized PBS and count cell numbers.
    7. Re-suspend cells at 5 x 105 cells/ml into complete αMEM media supplemented with 20 ng/ml of M-CSF.
    8. Culture cells at 5% CO2 and 37 °C overnight. Collect non-adherent cells and count their numbers and discard adherent cells (most of them are fibroblasts, mature macrophages and stromal cells). The expected yield of non-adherent cells is 5 x 106 cells/ml.
    9. For pit assay, incubate the cells with the bone slice as described in step A6.

  3. Bone resorption pit assay
    1. Label bone slices and sterilize them in a sterile hood with UV light the day before the experiment.
    2. On the day of the experiment, place bone slices in a 96 well (1 slice/well) labeled side down. Prepare bone marrow cells and plate at 50,000 cells/well or appropriate cell numbers and culture them under 200 µl of osteoclast differentiation media in the well.
    3. Cells are cultured for 10-14 days with feeding every 2-3 days. Culture media must be replenished completely during each feeding.
    4. Bone slices are then fixed with 2.5% glutaraldehyde in PBS for 30 min at room temperature.
    5. TRAP (Sidqui et al., 1995) stain cells and examine microscopically to observe TRAP (+) osteoclasts.
      Figure 2 shows an example of TRAP staining from tibia sections in 8-week old male mice. The three red arrows point to TRAP+ osteoclasts (which are stained red). A magnified image of an individual TRAP+ osteoclast is shown in the upper right hand corner.

      Figure 2. Image of osteoclasts positive for TRAP on the tibia section of an 8-week old male mouse. Tibia sections from 8-week old mice were stained with TRAP and counterstained with hematoxylin blue. Multinuclear osteoclasts appeared in red on trabecular bone surface (arrows). This representative example was taken from “SHP2 regulates osteoclastogenesis by promoting preosteoclasts fusion”, which is published in The FASEB Journal (Zhou et al., 2015).

    6. Remove cells from bone slice by sonicating 5-15 min in distilled water at 50-60 Hz to dislodge the cells from the bone slice.
      Note: Sonicate bone slices individually in glass beaker in order to prevent misidentification of the bone slices if identification numbers come off.
    7. Re-number the bone slices and completely dry bones on Whatman filter paper before staining.
    8. Prepare 1% toluidine blue the day before experiment.
    9. Stain bones 4 min total on 1% toluidine blue in 1% sodium borate. Stain by placing droplet (20 µl) of stain onto a Petri dish. Carefully place the bone slice onto the side of the droplet so that the slice is leaning onto the surface of the stain and not submerged under the surface of the drop. In other words, the bone slice should not break the surface tension of the droplet and get submerged by the droplet; once placed in a leaning position on the surface of the droplet, it should remain on the surface of the droplet.
      Two images of the result of step C9 are shown below in Figure 3. In each image, the bone slice is leaning on the 1% toluidine blue in 1% sodium borate droplet. The bone slice in each picture is not submerged under the 1% toluidine blue in 1% sodium borate droplet.

      Figure 3. Illustrative example of the proper position of bone slice on toluidine stain. A droplet of toluidine stain was pipetted on a microscope slide. Using tweezers, the bone slice was placed onto the surface of the toluidine stain. As shown in Figure 3, the proper configuration of the bone slice is on the surface of the toluidine stain droplet.

    10. Remove the bone slice from the droplet stain, and rinse by swirling bone slice in distilled water. Air dry and examine with light microscope.
      1. Use Olympus scope and turn on light source from box on left so that the light source comes from the top of the scope.
    11. Take pictures under microscope and evaluate the number and size of pit formation.
      Note: Pits should be stained in the color of toluidine.

Representative data

The following three representative examples have been taken from Zhou et al. (2015).

Figure 4. Illustrative examples of positive results of pit assays. The pit assay experiments shown above were conducted on dentin slices co-cultured with osteoclasts for 10 days in vitro. The pit assay results in 4A and 4B were generated without the addition of an extraneous SHP2 inhibitor. In 4C, however, SHP2 was inhibited with NSC-87877 during the co-culture process to block mature osteoclast formation (Zhou et al., 2015). Hence, a lack of mature osteoclasts was induced, which caused a lack of resorption pits as shown in 4C. Scale bar, 100 µm.

The “pits” created by the pit assay procedure are the blue areas in Figure 4A, Figure 4B, and Figure 4C. The red arrows in Figure 4A, 4B and 4C point to the pits created. The blue staining is due to the toluidine blue used in the procedure. The samples represented in Figure 4A and 4B both have a decent amount of pits. The sample represented in figure 4C has a relative absence of pits.
The mice strain used in the original experiment is the C57BL/6 strain.


  1. RBC lysis buffer
    0.747% NH4Cl
    0.017% Tris-HCl
  2. Osteoclast precursor culture media
    αMEM supplemented with 1% penicillin/streptomycin (P/S), 10% FBS (heat-inactivated) and 20 ng/ml of M-CSF
  3. Osteoclast differentiation media
    Osteoclast precursor culture media supplemented with 100 ng/ml RANKL
  4. 1% toluidine blue
    1% toluidine blue is prepared by combining 1 g sodium borate 10-hydrate in 100 ml dH2O.
    Dissolve 1 g toluidine blue in this solution and allow it to stand overnight
    Filter staining solution using Whatman filter paper and store at room temperature
  5. Glutaraldehyde solution
    Glutaraldehyde solution is 25% in H2O
    Dilute to 2.5% with ddH2O for use


This protocol was adapted from the previously published paper Zhou et al. (2015). This work was supported in part by the U. S. National Institutes of Health (NIH) National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R21AR57156 (to W. Y.) and NIH National Institute of General Medical Sciences Grant P20 GM103468. This study was also aided by a grant from the Pediatric Orthopaedic Society of North America and the Orthopaedic Research and Education Foundation (to W. Y.), and the U. S. Department of Agriculture Research Service program Grant #5450-51000- 046-00D (to J. C.).


  1. Cao, J., Wu, Q., Qin, Y. X., Reginato, A. M., Ehrlich, M. G. and Yang, W. (2015). SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion. FASEB J 29(5): 1635-1645.
  2. Charles, J. F. and Aliprantis, A. O. (2014). Osteoclasts: more than 'bone eaters'. Trends Mol Med 20(8): 449-459.
  3. Ross, F. P. (2006). M-CSF, c-Fms, and signaling in osteoclasts and their precursors. Ann N Y Acad Sci 1068: 110-116.
  4. Sidqui, M., Collin, P., Vitte, C. and Forest, N. (1995). Osteoblast adherence and resorption activity of isolated osteoclasts on calcium sulphate hemihydrate. Biomaterials 16(17): 1327-1332.
  5. Teitelbaum, S. L. (2000). Bone resorption by osteoclasts. Science 289(5484): 1504-1508.
  6. Zhou, Y., Mohan, A., Moore, D. C., Lin, L., Zhou, F. L., Cao, J., Wu, Q., Qin, Y. X., Reginato, A. M., Ehrlich, M. G. and Yang, W. (2015). SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion. FASEB J 29(5): 1635-1645.


尽管可以使用抗酒石酸盐的酸性磷酸酶(TRAP)染色来辅助鉴定破骨细胞,但是需要单独的方法来确定破骨细胞的骨吸收活性。 由于破骨细胞在骨基质吸收之后离开"凹陷"(Charles等人,2014),因此可以将斑点染色作为测量破骨细胞骨吸收活性的方法。 坑测定方案使研究人员能够将与破骨细胞与甲苯胺蓝共培养的骨切片染色,以便基于坑的数量,尺寸和深度可视化,捕获和分析破骨细胞再吸收活性(Zhou等人, et al。,2015)。 坑测定方案分为三个连续阶段:骨切片的制备(1); 制备破骨细胞前体(Ross等人,2006; Teitelbaum等人,2000)(2)和骨吸收坑测定(3)。

关键字:破骨细胞功能, 骨吸收, 坑试验


  1. parafilm
  2. 剃刀刀片
  3. 组织培养管(标准15或50ml锥形TC管)
  4. 带25 G针的5 ml注射器
  5. 细胞过滤器(70μm)(Corning,目录号:352350)
  6. 细胞培养96孔板
  7. 组织培养皿
  8. Whatman滤纸(Sigma-Aldrich,目录号:WHA10347509)
  9. 6-至10周龄的小鼠
  10. 磷酸盐缓冲盐水(PBS),灭菌,pH 7.4(Thermo Fisher Scientific,目录号:10010023)
  11. Dulbecco改良的Eagle培养基,高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  12. 青霉素/链霉素(10,000U/ml)(Thermo Fisher Scientific,目录号:15140-122)
  13. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:16000044)
  14. MEMα,核苷(Thermo Fisher Scientific,目录号:12571-063)
  15. 巨噬细胞刺激因子(M-CSF)(PeproTech,目录号:315-02)和受体激活的核因子k-B配体(RANKL)(PeproTech,目录号:315-11)
  16. 牛股骨(来自肉类市场)
  17. 乙醇
  18. 戊二醛(Sigma-Aldrich,目录号:G5882)
  19. 甲苯胺蓝(Sigma-Aldrich,目录号:T3260-5G)
  20. MilliQ水
  21. 酸性磷酸酶,白细胞(TRAP)试剂盒(Sigma-Aldrich,目录号:387A-1KT)
  22. 硼酸钠10水合物(Fisher Scientific,目录号:02-003-999)
  23. 台盼蓝(Thermo Fisher Scientific,目录号:T6146-5G)
  24. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434-500G)
  25. 盐酸盐(Tris-HCl)(Sigma-Aldrich,目录号:T5941-100G)
  26. 红细胞(RBC)裂解缓冲液(Sigma-Aldrich,目录号:R7757)(参见配方)
  27. 破骨细胞前体培养基(参见配方)
  28. 破骨细胞分化培养基(参见配方)
  29. 1%甲苯胺蓝(见配方)
  30. 戊二醛溶液(见配方)


  1. 镊子剪刀
  2. 钢锯
  3. Scalpel
  4. 水超声仪
  5. Isomet低速锯(BUEHLER,目录号:111280),带有切片刀片
  6. 光学显微镜


  1. 制备骨切片
    1. 将牛股骨的骨干横向切割成具有钢锯的2-3cm圆柱体,如图1A所示。
    2. 将圆柱体切成三段,将骨髓提起,如图1B所示。 任何粘连的肌肉,骨髓或骨膜用手术刀清除

      图1.牛股骨的横切和节段切割图。 A。牛股骨的图像,其描绘横向切成2-3cm圆柱体的骨干部分; B.(A)中提到的揉合圆柱体进一步被切成如图所示的三段(箭头)
    3. 然后用温和的水用少量洗涤剂将该片段超声处理几次,直到骨头是干净的。重复两到三次,直到骨头清洁。在原始实验中,将区段在50-60Hz下超声处理2分钟。
    4. 骨髓段用手术刀最终清洁任何粘附的组织,包裹在石蜡膜中并在冷冻条件(-20℃)下储存。来自相同圆柱体的段被给予相同的标签。
    5. 为了做出4.4×4.4×0.2mm的骨切片,用Isomet低速锯用晶片刀片将骨段横向切割成200μm厚的块。这些200微米切片用于生产最终的4.4 x 4.4 x 0.2毫米的骨切片
    6. 然后夹住200μm切片的叠层,并制成条带为4.4mm宽的切片。
    7. 将骨切片存储在70%乙醇中,直到使用。

  2. 制备骨髓衍生的破骨细胞前体
    1. 在无菌条件下从6至10周龄小鼠解剖股骨和胫骨,并将其置于冷PBS中。 用镊子剪刀和剃须刀刀片清除所有软组织,例如肌肉,脂肪,等。
    2. 用无菌PBS清洗股骨和胫骨两次,切两端
    3. 用5ml注射器(25G针)和DMEM(补充P/S和1%FBS)从两端将骨髓细胞冲洗到组织培养管(标准15或50ml锥形TC管)中。需要约40ml DMEM(补充有P/S和1%FBS)冲洗骨髓细胞。
    4. 通过在室温下离心637×g 5分钟来沉淀骨髓细胞并弃去上清液;在室温下将细胞重悬浮于3ml红细胞(RBC)裂解缓冲液中3-4分钟以破坏红细胞。
    5. 向细胞中加入7mlαMEM培养基(含10%FBS)以中和RBC缓冲液的作用;将细胞通过一个70微米的细胞过滤器进入一个50ml锥形管
    6. 通过在室温下离心637×g 5分钟来沉淀细胞并弃去上清液;用10 ml无菌PBS洗涤细胞一次,计数细胞数
    7. 将5×10 5个细胞/ml的细胞再悬浮于补充有20ng/ml M-CSF的完全αMEM培养基中。
    8. 在5%CO 2和37℃下培养细胞过夜。收集非贴壁细胞并计数其数量并丢弃贴壁细胞(大多数是成纤维细胞,成熟巨噬细胞和基质细胞)。非粘附细胞的预期产量为5×10 6个细胞/ml。
    9. 对于坑测定,如步骤A6中所述,用骨切片孵育细胞
  3. 骨吸收坑测定
    1. 在实验前一天,在无菌罩中用紫外线标记骨切片并将其消毒
    2. 在实验当天,将骨切片置于96孔(1切片/孔)标记面朝下。 准备骨髓细胞和板在50,000细胞/孔或适当的细胞数量,并在200微升的破骨细胞分化培养基在井中培养。
    3. 将细胞培养10-14天,每2-3天喂养一次。 培养基必须在每次喂养期间完全补充。
    4. 然后将骨切片用2.5%戊二醛的PBS溶液在室温下固定30分钟
    5. TRAP(Sidqui等人,1995)染色细胞并显微镜检查以观察TRAP(+)破骨细胞。
      图2显示了来自8周龄雄性小鼠的胫骨切片的TRAP染色的实例。三个红色箭头指向TRAP +破骨细胞(其染成红色)。单个TRAP +破骨细胞的放大图像显示在右上角。

      图2.在8周龄雄性小鼠的胫骨部分上TRAP阳性的破骨细胞的图像。 来自8周龄小鼠的胫骨切片用TRAP染色,并用苏木精蓝复染。多核破骨细胞出现在小梁骨表面上的红色(箭头)。该代表性实例取自"FASEB Journal(Zhou等人,2015)"中公开的"SHP2通过促进成骨细胞前体融合来调节破骨细胞形成"。

    6. 通过在50-60Hz的蒸馏水中超声处理5-15分钟从骨切片中去除细胞从骨切片中去除细胞。
    7. 在染色前重新对骨切片重新编号,并在Whatman滤纸上完全干燥骨。
    8. 在实验前一天准备1%甲苯胺蓝。
    9. 在1%硼酸钠中的1%甲苯胺蓝上染色骨总共4分钟。通过将微滴(20μl)的染色剂置于培养皿上而染色。小心地将骨切片放置在液滴的一侧,使切片倾斜到染色剂的表面上,而不是浸没在液滴的表面下。换句话说,骨切片不应该破坏液滴的表面张力并且被液滴浸没;一旦放置在液滴表面上的倾斜位置,它应该保留在液滴的表面上 步骤C9的结果的两个图像显示在下面的图3中。在每个图像中,骨切片倾向于在1%硼酸钠液滴中的1%甲苯胺蓝。每张图片中的骨切片不浸没在1%甲苯胺蓝的1%硼酸钠液滴中。

      图3.骨切片在甲苯胺染色上的适当位置的说明性实例。 在显微镜载玻片上吸取甲苯胺染色液滴。使用镊子,将骨切片置于甲苯胺染色剂的表面上。如图3所示,骨切片的适当构型在甲苯胺染色液滴的表面上
    10. 从液滴染色去除骨切片,并通过旋转骨切片在蒸馏水中冲洗。空气干燥并用光学显微镜检查。
      1. 使用Olympus示波器,打开左侧方框的光源,使光源来自示波器的顶部。
    11. 在显微镜下拍照并评估凹坑形成的数量和大小 注意:坑应该用甲苯胺的颜色染色。




由坑测定程序产生的"坑"是图4A,图4B和图4C中的蓝色区域。图4A,4B和4C中的红色箭头指向所创建的凹坑。蓝色染色是由于在该过程中使用的甲苯胺蓝。图4A和4B中表示的样本都具有相当大量的凹坑。图4C中所示的样品具有相对缺乏凹坑 在原始实验中使用的小鼠品系是C57BL/6品系。


  1. RBC裂解缓冲液
    0.747%NH 4 Cl
  2. 破骨细胞前体培养基
    补充有1%青霉素/链霉素(P/S),10%FBS(热灭活的)和20ng/ml M-CSF的αMEM。
  3. 破骨细胞分化培养基
    补充有100ng/ml RANKL的破骨细胞前体培养基
  4. 1%甲苯胺蓝
    通过在100ml dH 2 O中混合1g硼酸钠10水合物制备1%甲苯胺蓝。
  5. 戊二醛溶液
    戊二醛溶液在H 2 O中的浓度为25% 使用时用ddH 2 O稀释至2.5%


该协议改编自以前发表的论文Zhou等人。(2015)。这项工作部分由美国国家卫生研究院(NIH)国家关节炎和肌肉骨骼和皮肤疾病研究所授予R21AR57156(W.Y.)和NIH国家综合医学科学研究所Grant P20 GM103468部分支持。本研究还得到了北美儿科骨科学会和骨科研究与教育基金会(授予WY)和美国农业研究部服务计划授予#5450-51000-046-00D(授予JC)的资助, 。


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引用:Vesprey, A. and Yang, W. (2016). Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts. Bio-protocol 6(12): e1836. DOI: 10.21769/BioProtoc.1836.