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Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including blood vessels, kidney, articular cartilage, and cardiovascular tissue. Here, we describe the simple method for detecting and measuring the presence of mineralized nodules in cardiac ventricular fibroblasts by using von Kossa and alizarin red S staining, and a colorimetric method for calcium quantification, respectively.

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In vitro Biomineralization Assay
体外生物矿化试验

免疫学 > 免疫细胞染色
作者: Kyunghee Lee
Kyunghee LeeAffiliation: Department of Microbiology, Yeungnam University College of Medicine, Daegu, Korea
Bio-protocol author page: a1133
Minsuk Kwon
Minsuk KwonAffiliation: Department of Microbiology, Yeungnam University College of Medicine, Daegu, Korea
Bio-protocol author page: a1135
 and Daewon Jeong
Daewon JeongAffiliation: Department of Microbiology, Yeungnam University College of Medicine, Daegu, Korea
For correspondence: dwjeong@ynu.ac.kr
Bio-protocol author page: a1136
Vol 4, Iss 3, 2/5/2014, 6311 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1036

[Abstract] Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including blood vessels, kidney, articular cartilage, and cardiovascular tissue. Here, we describe the simple method for detecting and measuring the presence of mineralized nodules in cardiac ventricular fibroblasts by using von Kossa and alizarin red S staining, and a colorimetric method for calcium quantification, respectively.
Keywords: Calcification(钙化), Osteoblast(成骨细胞), Vascular smooth muscle cell(血管平滑肌细胞)

[Abstract]

Materials and Reagents

  1. Cardiac ventricular fibroblasts isolated from neonatal Sprague-Dawley rats by enzymatic dissociation (Lee et al., 2013)
    Note: Our protocol can be used with various cell types such as osteoblasts and vascular smooth muscle cells.
  2. Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and 4 mM L-glutamine (Hyclone, catalog number: SH 30243.01 )
  3. Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH 30919.03 )
  4. Inorganic phosphate (Pi) (pH 7.4)
  5. DPBS without Ca2+ and Mg2+ (Hyclone, catalog number: SH 30028.02 )
  6. Trypsin/EDTA (Hyclone, catalog number: SH 30042.01 )
  7. 70% ethanol
  8. Distilled deionized water (DDW)
  9. 0.6 N HCl solution
  10. Lysis solution
  11. 0.1 N NaOH
  12. 0.1% sodium dodecyl sulphate (SDS)
  13. QuantiChrome Calcium Assay Kit (BioAssay Systems, catalog number: DICA-500 )
  14. Working reagent (refer to QuantiChrome Calcium Assay Kit manual for detail) (BioAssay Systems, catalog number: DICA-500) (see Reference 2)
  15. Bio-Rad DC protein assay kit (Bio-Rad Laboratories, catalog number: 500-0016 )
  16. Diluted protein standards (e.g., 0, 2, 4, 6, 8, 12, 16 and 20 mg/dl)
  17. 100 ng/ml receptor activator of NF-kB ligand (RANKL) (Sigma-Aldrich, catalog number: R0525 )
  18. 5% Aqueous silver nitrate solution (Sigma-Aldrich, catalog number: S7279 ) (see Recipes)
  19. 5% sodium thiosulfate (Sigma-Aldrich, catalog number: S7026 ) (see Recipes)
  20. 2% alizarin red S solution (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
  21. 1 M NaH2PO4 (pH 7.4) (Sigma-Aldrich, catalog number: S6566 ) (see Recipes)

Equipment

  1. 48-well plate
  2. 96-well plate
  3. Spectrophotometer or 96-well reader
  4. 37 °C, 5% CO2 cell culture incubator
  5. Inverted microscope
  6. Aspirator
  7. UV-visualizer
  8. High-watt lamp (60-100 watt)

Procedure

  1. Detection of calcium deposits
    1. Von Kossa Staining
      1. Cardiac ventricular fibroblast cells (2 x 104 cells/well in 48-well plates) are cultured in 500 μl of DMEM containing 10% FBS in the absence or presence of 3 mM inorganic phosphate (Pi; pH 7.4) for 6 days in 37 °C, 5% CO2  cell culture incubator for inducing mineralization. The media are freshly changed every 2 days.
        Note: For inducing ectopic calcification in cardiac fibroblasts by high concentration of inorganic phosphate.
      2. The cultured cells are rinsed twice with 250 μl DPBS and fixed in 250 μl of ice-cold 70% ethanol for 1 h at room temperature without any agitation. 
      3. After fixation, DPBS and ethanol are removed and rinse with DDW carefully.
      4. After aspirating DDW on top of the fixed cells, add 250 μl of 5% silver nitrate solution and place the cell container in bright light such as sunlight, UV light or high-watt lamp until calcium deposits turns proper black (or dark brown).
      5. Rinse in 3 changes of 250 μl DDW briefly by gentle shaking.
      6. Rinse un-reacted silver with 250 μl of 5% sodium thiosulfate and keep for 5 min at room temperature (optional).
      7. Rinse in 3 changes of 250 μl DDW briefly by gentle shaking.
    2. Alizarin red S staining
      1. Fix the cultured cells with 250 μl of ice-cold 70% ethanol for 1 h at room temperature.
      2. After 3 changes of washing with 250 μl of DDW, stain the cells with 250 μl of 2% alizarin red S stain solution for 30 to 60 min at room temperature.
      3. Rinse in 2 changes with 250 μl of DDW.
      4. After removal of unincorporated excess dye with DDW, the mineralized nodules were stained as red spots.

  2. Calcium quantification
    Calcium content of the supernatants is determined colorimetrically with use of the QuantiChrome Calcium Assay Kit as described by manufacturer’s instructions. Briefly,
    1. Cultured cells are washed twice with 250 μl of DPBS and decalcified with 250 μl of 0.6 N HCl for 12 h. 
    2. Prepare working reagent by mixing equal volume of reagent A and B. Equilibrate to room temperature before use.
    3. Transfer 5 μl of diluted standards or samples into each wells of a clear bottom 96-well plate.
    4. Add 200 μl of working reagent and tap lightly to mix.
    5. After incubation for 3 min at room temperature, read the absorbance at 570-650 nm with 96-well reader.
    6. The remaining cells are washed three times with 250 μl of DPBS and solubilized in 200 μl of lysis solution containing 0.1 N NaOH and 0.1% sodium dodecyl sulphate (SDS) at room temperature for 5 min.
    7. The protein content is measured with a Bio-Rad DC protein assay kit.
    8. Calcium content is normalized to the total protein content of the whole cells (μg of Ca2+/mg protein of whole cells, see Figure 1).


      Figure 1. The synergic effect of RANKL on Pi-induced cardiac fibroblast calcification (Lee et al., 2013). Primary cardiac fibroblast cells were treated with 3 mM Pi in the absence or presence of 100 ng/ml RANKL for 3 d. Calcium content in cells was measured, and calcium deposits were visualized by von Kossa staining. A representative image is presented in the top panel. Scale bar, 100 μm (upper panel). Calcium contents were measured by QuantiChrome Calcium Assay kit (lower panel). Quantitative data are means ± SD (n = 3).

Recipes

  1. 5% Aqueous silver nitrate solution
    Dissolve 5 g of silver nitrate in 100 ml DDW
  2. 5% sodium thiosulfate
    Dissolve 5 g of sodium thiosulfate in 100 ml DDW
  3. 2% alizarin red S solution
    Dissolve 2 g of alizarin red S (sodium alizarin sulphonate) in 100 ml DDW and the pH was adjusted to 4.1-4.3 using 0.5% ammonium hydroxide.
  4. 1 M NaH2PO4 (pH 7.4)
    Dissolve 1.2 g of NaH2PO4 in 10 ml DDW and the pH is adjusted to 7.4 using NaOH.

Acknowledgments

This protocol was adapted from the previously published report Lee et al. (2013) and the QuantiChrome Calcium Assay Kit manual.

References

  1. Lee, K., Kim, H., Park, H. S., Kim, K. J., Song, H., Shin, H. I., Kim, H. S., Seo, D., Kook, H., Ko, J. H. and Jeong, D. (2013). Targeting of the osteoclastogenic RANKL-RANK axis prevents osteoporotic bone loss and soft tissue calcification in coxsackievirus B3-infected mice. J Immunol 190(4): 1623-1630.
  2. QuantiChrome Calcium Assay Kit manual. http://www.bioassaysys.com/file_dir/DICA.pdf.

材料和试剂

  1. 通过酶解离从新生Sprague-Dawley大鼠分离的心室心室成纤维细胞(Lee等人,2013)
    注意:我们的方案可用于各种细胞类型,如成骨细胞和血管平滑肌细胞。
  2. 具有高葡萄糖和4mM L-谷氨酰胺(Hyclone,目录号:SH 30243.01)的Dulbecco改良的Eagle培养基(DMEM)
  3. 胎牛血清(FBS)(Hyclone,目录号:SH30919.03)
  4. 无机磷酸盐(Pi)(pH 7.4)
  5. DPBS,不含Ca 2+和Mg 2+ 2+(Hyclone,目录号:SH 30028.02)。
  6. 胰蛋白酶/EDTA(Hyclone,目录号:SH30042.01)
  7. 70%乙醇
  8. 蒸馏去离子水(DDW)
  9. 0.6 N HCl溶液
  10. 裂解液
  11. 0.1 N NaOH
  12. 0.1%十二烷基硫酸钠(SDS)
  13. QuantiChrome钙测定试剂盒(BioAssay Systems,目录号:DICA-500)
  14. 工作试剂(详见QuantiChrome Calcium Assay Kit手册)(BioAssay Systems,目录号:DICA-500)(参见参考文献2)
  15. Bio-Rad DC蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0016)
  16. 稀释的蛋白质标准品(例如,0,2,4,6,8,12,16和20 mg/dl)
  17. 100ng/ml NF-kB配体的受体激活剂(RANKL)(Sigma-Aldrich,目录号:R0525)
  18. 5%硝酸银水溶液(Sigma-Aldrich,目录号:S7279)(参见Recipes)
  19. 5%硫代硫酸钠(Sigma-Aldrich,目录号:S7026)(参见Recipes)
  20. 2%茜素红S溶液(Sigma-Aldrich,目录号:A5533)(参见Recipes)
  21. 1M NaH 2 PO 4(pH 7.4)(Sigma-Aldrich,目录号:S6566)(参见配方)。

设备

  1. 48孔板
  2. 96孔板
  3. 分光光度计或96孔读数器
  4. 37℃,5%CO 2细胞培养箱中培养
  5. 倒置显微镜
  6. 吸气器
  7. 紫外可视化仪
  8. 高功率灯(60-100瓦)

程序

  1. 钙沉积物的检测
    1. Von Kossa染色
      1. 在500μl含有10%FBS的DMEM中,在不存在或存在3mM无机磷酸盐(Pi; pH值)的情况下培养心室心室成纤维细胞(在48孔板中2×10 4个细胞/7.4)在37℃,5%CO 2细胞培养箱中培养6天,用于诱导矿化。 介质每2天更新一次。
        注意:用于通过高浓度的无机磷酸盐诱导心脏成纤维细胞中的异位钙化
      2. 将培养的细胞用250μlDPBS冲洗两次,并在室温下固定在250μl冰冷的70%乙醇中1小时,无需任何搅拌。
      3. 固定后,取出DPBS和乙醇,并仔细用DDW冲洗。
      4. 在固定细胞顶部吸入DDW后,加入250μl5%硝酸银溶液,将细胞容器置于明亮的光照下,如阳光,紫外光或高瓦灯,直到钙沉积物变成适当的黑色(或深棕色)。
      5. 轻轻振荡,轻轻冲洗3次250μlDDW。
      6. 冲洗未反应的银与250微升5%硫代硫酸钠,并在室温下保持5分钟(可选)。
      7. 轻轻振荡,轻轻冲洗3次250μlDDW。
    2. 茜素红S染色
      1. 固定培养细胞与250微升冰冷的70%乙醇在室温下1小时。
      2. 用250μlDDW洗涤3次后,用250μl2%茜素红染色溶液在室温下染色细胞30至60分钟。
      3. 用250μlDDW冲洗2次。
      4. 用DDW除去未掺入的过量染料后,矿化结节被染成红色斑点
  2. 钙定量
    使用如制造商说明书所述的QuantiChrome钙测定试剂盒比色测定上清液的钙含量。 简而言之,
    1. 培养的细胞用250μlDPBS洗涤两次,并用250μl0.6N HCl脱钙12小时。
    2. 通过混合等体积的试剂A和B制备工作试剂。使用前平衡至室温。
    3. 将5μl稀释的标准品或样品转移到透明底部96孔板的每个孔中。
    4. 加入200微升工作试剂,轻轻点击混合。
    5. 在室温下孵育3分钟后,用96孔读数器读取570-650nm的吸光度。
    6. 剩余的细胞用250μlDPBS洗涤三次,并在室温下溶解在200μl含有0.1N NaOH和0.1%十二烷基硫酸钠(SDS)的裂解溶液中5分钟。
    7. 用Bio-Rad DC蛋白测定试剂盒测定蛋白质含量。
    8. 钙含量相对于全细胞的总蛋白质含量(μgCa 2+/mg/mg蛋白质的全细胞,参见图1)标准化。


      图1. RANKL对Pi诱导的心脏成纤维细胞钙化的协同效应(Lee 等 ,2013)强>在缺乏或存在100ng/ml RANKL的情况下用3mM PI处理原代心脏成纤维细胞3天。测量细胞中的钙含量,通过von Kossa染色观察钙沉积物。代表性图像显示在顶部面板中。比例尺,100μm(上图)。通过QuantiChrome Calcium Assay试剂盒(下图)测量钙含量。定量数据为平均值±SD(n = 3)。

食谱

  1. 5%硝酸银水溶液
    将5g硝酸银溶于100ml DDW中
  2. 5%硫代硫酸钠
    将5g硫代硫酸钠溶于100ml DDW中
  3. 2%茜素红S溶液
    将2g茜素红S(茜素磺酸钠)溶解在100ml DDW中,用0.5%氢氧化铵将pH调节至4.1-4.3。
  4. 1 H NaH 2 PO 4(pH 7.4)
    在10ml DDW中溶解1.2g NaH 2 PO 4,并使用NaOH将pH调节至7.4。

致谢

此协议改编自先前发表的报告Lee等人(2013)和QuantiChrome Calcium Assay Kit手册。

参考文献

  1. Lee,K.,Kim,H.,Park,HS,Kim,KJ,Song,H.,Shin,HI,Kim,HS,Seo,D.,Kook,H.,Ko,JHand Jeong, 2013)。 靶向破骨细胞RANKL-RANK轴可预防柯萨奇病毒B3感染的骨质疏松性骨质流失和软组织钙化 小鼠。 J Immunol 190(4):1623-1630。
  2. QuantiChrome钙测定试剂盒手册。 http://www.bioassaysys.com/file_dir/DICA.pdf
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How to cite this protocol: Lee, K., Kwon, M. and Jeong, D. (2014). In vitro Biomineralization Assay. Bio-protocol 4(3): e1036. DOI: 10.21769/BioProtoc.1036; Full Text



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11/8/2014 5:09:20 AM  

Naif Almontashiri
University of Ottawa Heart Institute

Hi,

For Calcium quantification, Do I have to add the decalcifying solution, 0.1 N HCL, on cultured cells after washing with DPBS? Can you tell me more or send me your decalcification protocol? The 12 hours incubation at what temperature? do i have to incubate it on a rotator or shaker?

11/13/2014 6:14:10 PM  

Daewon Jeong (Author)
Department of Microbiology, Yeungnam University College of Medicine, Korea

To quantify calcium, cultured cells need to be washed twice with PBS and decalcified with 0.6 N HCl for 12 h under room temperature. You do not need to incubated cells on a rotator or shaker.

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