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Binding to Secreted Bone Matrix in vitro
分泌骨基质结合的体外试验   

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本实验方案简略版
The Journal of Immunology
Mar 2013

 

Abstract

This method examines the bone matrix binding capacity of proteins. Using osteogenic differentiation medium, multipotent stromal cells (MSC) are induced to differentiate into osteocytes in vitro and to secrete bone matrix. The latter is confirmed using Alizarin red S staining, which detects the presence of calcific deposits (hydroxyapatite). These calcific deposits are used to test the bone binding properties of proteins. The binding to the calcific deposits is assessed by Western blot analysis.

Materials and Reagents

  1. Multipotent stromal cells (MSC) isolated from mouse bone marrow [see protocol “Isolation of Multipotent Stromal Cells from Mouse Bone Marrow” (Tormo et al., 2014)]
  2. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL )
  3. Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL )
  4. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 12483 )
  5. 0.53 mM 0.05% Trypsin/EDTA (Wisent, catalog number: 325-042-EL )
  6. PBS without Ca2+ and Mg2+ (Wisent, catalog number: 311-01-CL )
  7. HEPES (Wisent, catalog number: 330-050-EL )
  8. 10 nM Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
  9. 50 μM Ascorbic acid-2 phosphate (vitamin C) (Sigma-Aldrich, catalog number: A4403 )
  10. 50 nM Cholecalciferol (Vitamin D3) (Sigma-Aldrich, catalog number: C9756 )
  11. 10 mM B-glycerophosphate (Sigma-Aldrich, catalog number: G9891 )
  12. 100x Protease and phosphatase inhibitors (Thermo Fisher Scientific, Pierce, catalog number: PI78447 )
  13. IL-27 (1 μg) (R&D Systems, catalog number: 2799-ML-010/CF ) or any recombinant protein to be tested
  14. Bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Pierce, catalog number: PI23225 )
  15. Distilled water
  16. Ethanol
  17. Anti-IL-27 p28 biotinylated antibody (R&D Systems, catalog number: BAF1834 ) or biotinylated antibody specific for the protein to be tested
  18. Streptavidin-HRP (Thermo Fisher Scientific, Pierce, catalog number: PI21130 )
  19. SDS-PAGE loading buffer without bromophenol blue or DTT
  20. Urea buffer (Fisher Scientific, catalog number: BP169-10 )
  21. 1 M Sodium phosphate monobasic (Fisher Scientific, catalog number: BP330-1 )
  22. 1 M Sodium phosphate dibasic (Fisher Scientific, catalog number: BP332-1 )
  23. TBS-Tween
  24. 3% BSA
  25. Dexamethasone solution (see Recipes)
  26. 10x Glycerophosphate solution (see Recipes)
  27. 1,000x Vitamin C solution (see Recipes)
  28. Osteogenic differentiation medium (50 ml) (see Recipes)
  29. Alizarin red S (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
  30. Urea/Phosphate lysis buffer (see Recipes)

Equipment

  1. 15 ml tube
  2. Centrifuge for cell culture
  3. 6 well tissue culture plates (BD Biosciences, catalog number: DL-353046 )
  4. 96 well tissue culture plates with flat bottoms (BD Biosciences, catalog number: DL-353072 )
  5. Inverted phase-contrast microscope
  6. 37 °C, 5% CO2 cell culture incubator
  7. Cell scrapers (Corning, catalog number: 3010 )
  8. Microplate reader for BCA (562 nm)
  9. SDS-PAGE Equipment
  10. Western blot Equipment

Procedure

  1. MSC are rinsed gently one time with 2 ml of PBS and trypsinized. For cells trypsinization: add 0.5 ml of trypsin on the rinsed cells. Wait 3 to 5 min until cells peel off the plastic surface. Transfer cells in a 15 ml tube and add quickly 10 ml of DMEM containing 10 % FBS in order to inhibit trypsin action.
  2. The cells are pelleted at 435 x g for 5 min at 4 °C.
  3. The supernatant is removed and the cells are incubated in 6 wells plate at a density of 5,000-10,000 cells per well, in 2 ml of DMEM complemented with 10% FBS.
  4. The cells are incubated in the 5% CO2 incubator at 37 °C overnight.
  5. MSC are washed twice with PBS by adding and removing gently 2 ml of PBS per well without any agitation. Cells must be still adherent to the bottom of wells after washes. The adherent cells are then incubated with osteogenic differentiation medium.
  6. The osteogenic differentiation medium is changed every 2 days for 10 days.
  7. At day 10, the differentiated cells are washed with PBS and exposed to Alizarin red S preparation for 5 min at room temperature without any agitation.
  8. Cells are rinsed with distilled water and the red coloration indicating an osteogenic differentiation is assessed by microscopy (see Figure 1).


    Figure 1. Multipotent stromal cells were differentiated into osteocytes for 10 d. Osteocyte differentiation was detected by Alizarin red S staining (Tormo et al., 2013). (Copyright 2013. The American Association of Immunologists, Inc)

  9. Each well is incubated with 1 μg IL-27 (or other molecules being tested) in 500 μl serum-free DMEM for 1 h on ice without agitation (verify that all the well is recovered by medium). DMEM alone is used as negative control.
  10. Cells are washed 5 times with 1 ml of ice-cold serum-free DMEM without agitation and scraped in 100 μl urea/phosphate lysis buffer supplemented with protease and phosphatase inhibitors.
  11. Lysates are transferred in 1.5 ml tubes and diluted 10 times with bromophenol blue-free and DTT-free SDS-PAGE loading buffer.
  12. Protein concentration is quantified using a BCA kit (follow manufacturer protocol) and 50 μg of the total protein is analysed by SDS-PAGE (after adding 5% b-mercaptoethanol to each tubes) and Western blot.
  13. IL-27 is detected after blocking the Western blot membrane 30 min at room temperature in TBS-Tween containing 3% BSA followed by successive incubation with biotinylated goat anti-mouse p28 Ab (incubated over-night at 4 °C) and HRP-labelled streptavidin (incubated 1 h at room temperature). All incubations are performed in TBS-Tween containing 3% BSA. Washes are performed following incubation with Ab or streptavidin. These membrane washes consist of 3 incubations of 10 min at room temperature in 20 ml of TBS-Tween with agitation.

Recipes

  1. 1,000x Dexamethasone solution
    Dissolve 1.2 mg dexamethasone in 1,223 μl ethanol
    Transfer 10 μl of this 2.5 x 10-3 M solution to 2.5 ml DMEM
    This solution is now 1x10-5 M (1,000x)
  2. 10x Glycerophosphate solution
    Dissolve 630 mg b-glycerophosphate in 20 ml DMEM/15 mM HEPES (pH 7.5)
    Filter through a 0.22 μm pore size membrane under the tissue culture hood
  3. 1,000x Vitamin C solution
    Dissolve 50 mg vitamin C in 10 ml DMEM/15 mM HEPES (pH 7.5)
    Filter through a 0.22 μm pore size membrane under the tissue culture hood
  4. Osteogenic differentiation medium (50 ml)
    5 μl dexamethasone at 0.5 mM (1,000x)
    1 ml glycerophosphate at 500 mg/ml
    50 μl vitamin C at 50 mM (1,000x)
    50 μl vitamin D3 at 50 μM
    5 ml FBS
    Complete to 50 ml with DMEM
  5. Alizarin red S preparation
    Phosphate-buffered saline (PBS) with 2% Alizarin red S
  6. (8 M) Urea/Phosphate 400 mM lysis buffer (pH 7.5) (100 ml)
    Mix 77.4 ml of 1 M Na2HPO4 with 22.6 ml of 1 M NaH2PO4 to obtain 100 ml of 1 M phosphate buffer (pH 7.5)
    Mix 48 g Urea with 40 ml of 1 M phosphate buffer (pH 7.5)
    pH to 7.5 with Na2HPO4
    Add dH2O to 100 ml
    Note: Add 1x phosphatase and protease inhibitor (dilute the stock 1/100 in the desired volume of Urea/Phosphate solution to obtain 1x final concentration) just before using.

Acknowledgments

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

References

  1. Tormo, A. J., Beaupre, L. A., Elson, G., Crabe, S. and Gauchat, J. F. (2013). A polyglutamic acid motif confers IL-27 hydroxyapatite and bone-binding properties. J Immunol 190(6): 2931-2937.
  2. Tormo, A. J., Rafei, M. and Gauchat, J. F. (2014). Isolation of multipotent stromal cells from mouse bone marrow. Bio-protocol 4(4): e1031.

简介

这种方法检查蛋白质的骨基质结合能力。 使用成骨分化培养基,诱导多潜能基质细胞(MSC)在体外分化成骨细胞和分泌骨基质。 后者使用茜素红S染色证实,其检测钙化沉积物(羟基磷灰石)的存在。 这些钙沉积物用于测试蛋白质的骨结合性质。 通过蛋白质印迹分析评估与钙化沉积物的结合。

材料和试剂

  1. 从小鼠骨髓分离的多能基质细胞(MSC)[参见方案"多能性基质的分离 来自小鼠骨髓的细胞"(Tormo等人,2014)]
  2. Dulbecco's Modified Eagle's Medium具有稳定的L-谷氨酰胺(DMEM)的高葡萄糖(Wisent,目录号:319-015-CL)
  3. 青霉素/链霉素溶液(Wisent,目录号:450-201-EL)
  4. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:12483)
  5. 0.53mM 0.05%胰蛋白酶/EDTA(Wisent,目录号:325-042-EL)
  6. 没有Ca 2+和Mg 2+ 2 + (Wisent,目录号:311-01-CL)的PBS;
  7. HEPES(Wisent,目录号:330-050-EL)
  8. 10nM地塞米松(Sigma-Aldrich,目录号:D4902)
  9. 50μM抗坏血酸-2磷酸盐(维生素C)(Sigma-Aldrich,目录号:A4403)
  10. 50nM胆钙化醇(维生素D3)(Sigma-Aldrich,目录号:C9756)
  11. 10mM B-磷酸甘油(Sigma-Aldrich,目录号:G9891)
  12. 100x蛋白酶和磷酸酶抑制剂(Thermo Fisher Scientific,Pierce,目录号:PI78447)
  13. IL-27(1μg)(R& D Systems,目录号:2799-ML-010/CF)或任何待测试的重组蛋白质
  14. 二辛可宁酸测定(BCA)(Thermo Fisher Scientific,Pierce,目录号:PI23225)
  15. 蒸馏水
  16. 乙醇
  17. 抗IL-27 p28生物素化抗体(R& D Systems,目录号:BAF1834)或对于待测蛋白特异的生物素化抗体
  18. 链霉亲和素-HRP(Thermo Fisher Scientific,Pierce,目录号:PI21130)
  19. 不含溴酚蓝或DTT的SDS-PAGE上样缓冲液
  20. 尿素缓冲液(Fisher Scientific,目录号:BP169-10)
  21. 1 M磷酸二氢钠(Fisher Scientific,目录号:BP330-1)
  22. 1M磷酸氢二钠(Fisher Scientific,目录号:BP332-1)
  23. TBS-Tween
  24. 3%BSA
  25. 地塞米松溶液(参见配方)
  26. 10x甘油磷酸盐溶液(见配方)
  27. 1,000x维生素C溶液(见配方)
  28. 成骨分化培养基(50ml)(参见配方)
  29. 茜素红S(Sigma-Aldrich,目录号:A5533)(参见Recipes)
  30. 尿素/磷酸盐裂解缓冲液(见配方)

设备

  1. 15 ml管
  2. 离心机用于细胞培养
  3. 6孔组织培养板(BD Biosciences,目录号:DL-353046)
  4. 具有平底的96孔组织培养板(BD Biosciences,目录号:DL-353072)
  5. 反相相差显微镜
  6. 37℃,5%CO 2细胞培养箱中培养
  7. 细胞刮刀(Corning,目录号:3010)
  8. BCA(562 nm)微孔板读数器
  9. SDS-PAGE设备
  10. Western印迹设备

程序

  1. MSC用2ml PBS轻轻冲洗一次并用胰蛋白酶消化。 对于细胞胰蛋白酶化:在漂洗的细胞上加入0.5ml胰蛋白酶。 等待3到5分钟,直到细胞从塑料表面剥离。 转移细胞在15毫升管,并迅速加入10毫升含10%FBS的DMEM,以抑制胰蛋白酶作用。
  2. 细胞在4℃以435×g离心5分钟沉淀
  3. 除去上清液,将细胞在6孔板中以5,000-10,000个细胞/孔的密度在2ml补充有10%FBS的DMEM中孵育。
  4. 将细胞在5%CO 2培养箱中在37℃温育过夜
  5. 通过在没有任何搅拌的情况下每孔轻轻地添加和移除2ml PBS,用PBS洗涤MSC两次。 在洗涤后细胞必须仍然粘附到孔的底部。 然后将贴壁细胞与成骨分化培养基一起温育
  6. 成骨分化培养基每2天更换10天
  7. 在第10天,用PBS洗涤分化的细胞,并在室温下暴露于茜素红S制剂5分钟,无任何搅拌。
  8. 用蒸馏水冲洗细胞,通过显微镜检查评估表明成骨分化的红色(参见图1)。


    图1.多潜能基质细胞分化成骨细胞10天。通过茜素红S染色检测骨形态分化(Tormo等人,2013)。 (版权所有2013年。美国免疫学家协会)

  9. 将每个孔与500μl无血清DMEM中的1μgIL-27(或其他待测分子)一起在冰上孵育1小时,无需搅拌(确认所有孔通过培养基回收)。单独的DMEM用作阴性对照
  10. 将细胞用1ml冰冷的无血清DMEM洗涤5次,无需搅动,并在100μl添加了蛋白酶和磷酸酶抑制剂的尿素/磷酸盐裂解缓冲液中刮取。
  11. 将裂解物转移到1.5ml管中,用不含溴酚蓝和无DTT的SDS-PAGE上样缓冲液稀释10倍。
  12. 使用BCA试剂盒(遵循制造商方案)定量蛋白质浓度,通过SDS-PAGE(在每管加入5%b-巯基乙醇后)和Western印迹分析50μg总蛋白。
  13. 在室温下在含有3%BSA的TBS-Tween中封闭Western印迹膜30分钟,随后用生物素化的山羊抗小鼠p28Ab(在4℃孵育过夜)和HRP标记的链霉亲和素(在室温下温育1小时)。所有孵育在含有3%BSA的TBS-Tween中进行。在与Ab或链霉亲和素孵育后进行洗涤。这些膜洗涤由在室温下在20ml TBS-Tween中在搅拌下孵育10分钟组成。

食谱

  1. 1,000x地塞米松溶液
    将1.2mg地塞米松溶解在1,223μl乙醇中 将10μl的该2.5×10 -3 M溶液转移到2.5ml DMEM中 此解决方案现在为1x10 -5 M(1,000x)
  2. 10x甘油磷酸盐溶液
    将630mg b-甘油磷酸溶解在20ml DMEM/15mM HEPES(pH7.5)中 在组织培养罩下通过0.22μm孔径的膜过滤
  3. 1,000x维生素C溶液
    将50mg维生素C溶解在10ml DMEM/15mM HEPES(pH7.5)中 在组织培养罩下通过0.22μm孔径的膜过滤
  4. 成骨分化培养基(50ml)
    5微升地塞米松0.5mM(1,000x)
    1ml 500mg/ml的甘油磷酸盐 50μl50 mM维生素C(1,000x)
    50微升维生素D3 50微升
    5ml FBS
    用DMEM完成至50ml
  5. 茜素红S制剂
    具有2%茜素红S的磷酸盐缓冲盐水(PBS)
  6. (8M)尿素/磷酸盐400mM裂解缓冲液(pH7.5)(100ml) 将77.4ml 1M Na 2 HPO 4与22.6ml 1M NaH 2 PO 4溶液混合,得到 100ml的1M磷酸盐缓冲液(pH7.5) 将48g尿素与40ml 1M磷酸盐缓冲液(pH7.5)混合 用Na 2 HPO 4水溶液将pH调节至7.5 将dH <2> O添加到100 ml
    注意:在使用前加入1x磷酸酶和蛋白酶抑制剂(在所需体积的尿素/磷酸盐溶液中稀释1/100以获得1x最终浓度)。

致谢

该协议改编自Tormo等人(2013)。

参考文献

  1. Tormo,A.J.,Beaupre,L.A.,Elson,G.,Crabe,S.and Gauchat,J.F。(2013)。 聚谷氨酸模体赋予IL-27羟基磷灰石和骨结合特性。 J Immunol 190(6):2931-2937。
  2. Tormo,A.J.,Rafei,M。和Gauchat,J.F。(2014)。 从小鼠骨髓中分离多潜能基质细胞 生物协议 4 (4):e1031
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tormo, A. J., Beauséjour, C. and Gauchat, J. (2014). Binding to Secreted Bone Matrix in vitro. Bio-protocol 4(4): e1030. DOI: 10.21769/BioProtoc.1030.
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