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Isolation of Multipotent Stromal Cells from Mouse Bone Marrow
从小鼠骨髓中分离多能间质干细胞   

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Abstract

Generating mouse multipotent stromal cells (MSC) from bone-marrow cells is usefull for a wide range of applications. Effectively, these MSC can differentiate into adipocytes, osteocytes [See “Binding to Secreted Bone Matrix in vitro” (Tormo et al., 2014)] or chondrocytes upon culture in specific differentiation medium.

Materials and Reagents

  1. 6-8 weeks old mouse
  2. PBS without Ca2+ and Mg2+ (Wisent, catalog number: 311-01-CL )
  3. Dulbecco's Modified Eagle's Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL )
  4. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 12483 )
  5. Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL )
  6. Trypan blue (Life Technologies, Gibco®, catalog number: 15250-061 )
  7. Trypsin 0.05%/EDTA 0.53 mM (Wisent, catalog number: 325-042-EL )
  8. APC-conjugated anti-CD31 antibody (clone MEC13.3) (BD Biosciences, catalog number: 551262 )
  9. FITC-conjugated anti-CD45 antibody (clone 30-F11) (BD Biosciences, catalog number: 553080 )
  10. APC-conjugated anti-CD44 antibody (clone IM7) (BD Biosciences, catalog number: 559250 )
  11. PE-conjugated anti-CD105 antibody (clone MJ7/18) (BD Biosciences, catalog number: 562759 )
  12. FITC-conjugated anti-CD90 antibody (clone 5E10) (BD Biosciences, catalog number: 555595 )

Equipment

  1. Scissors and forceps
  2. Syringe 1cc with 27 Gauge x 1-½ needle (BD, catalog numbers: BD-309659 and BD-305109 )
  3. Petri dishes 100 x 20 mm (BD, catalog number: DL-353003 )
  4. 50 ml conical tubes (Progene®, catalog number: 71-5000-B )
  5. Table top centrifuge
  6. Culture hood
  7. Hemocytometer
  8. T-25 flask (BD, catalog number: 353108 )
  9. 37 °C, 5% CO2 Cell culture incubator
  10. Flow cytometer (e.g. BD LSRFortessa)

Procedure

  1. Under culture hood, flush tibia and femora from one 6-8 weeks old mouse with FBS-free DMEM in a dish with DMEM previously warm at 37 °C (see Video 1).

    Video 1. Mouse-bone marrow collection

  2. With a syringe and needle, aspirate and eject 2-3 times the bonne marrow to disrupt it.
  3. Transfer in a 50 ml conical tube.
  4. Centrifuge 10 min at 430 x g at 4 °C.
  5. Resuspend the pellet with DMEM supplemented with 15% FBS and Penicillin/Streptomycin at a density of 106 cells/ml.
  6. Plate 1 x 107 cells (10 ml) in a T25 flask.
  7. Incubate at 37 °C-5 % CO2 for 7-10 days without changing medium.
  8. Remove the supernatant in order to keep only adherent cells and wash the flask 2 times with 5 ml of PBS.
  9. Trypsinize cells for expansion. 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.
  10. After 7 - 10 days, assess the phenotype of the growing adherent cells by flow cytometry.
    1. Stain cells with fluorescein isothiocyanate (FITC)-labeled anti-CD45 (clone 30-F11) and with allophycocyanine (APC)-labeled anti-CD31 (clone MEC133). These antibody should be diluted 1/200. Cells should be negative for these two markers as CD45 is a hematopoietic cells marker, and CD31 is an endothelial cells marker.
    2. Stain cells with APC-labeled anti-CD44 (clone IM7), phycoerythrin (PE)-labeled anti-CD105 (clone MJ7/18) and with FITC-labeled anti-CD90 (clone 5E10), all used at a 1/200 dilution. Cells should be positive for these three markers.

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., Beauséjour, C. and Gauchat, J. F. (2014). Binding to secreted bone matrix in vitro. Bio-protocol 4(4): e1030.

简介

从骨髓细胞产生小鼠多潜能基质细胞(MSC)对于广泛的应用是有用的。 有效地,这些MSC可以分化成脂肪细胞,骨细胞[参见"体外与分泌的骨骼基质结合" (Tormo等人,2014)]或软骨细胞在特定分化培养基中培养。

材料和试剂

  1. 6-8周龄的老鼠
  2. 没有Ca 2+和Mg 2+ 2 + (Wisent,目录号:311-01-CL)的PBS;
  3. 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)
  4. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:12483)
  5. 青霉素/链霉素溶液(Wisent,目录号:450-201-EL)
  6. 台盼蓝(Life Technologies,Gibco ,目录号:15250-061)
  7. 胰蛋白酶0.05%/EDTA 0.53mM(Wisent,目录号:325-042-EL)
  8. APC结合的抗CD31抗体(克隆MEC13.3)(BD Biosciences,目录号:551262)
  9. FITC缀合的抗CD45抗体(克隆30-F11)(BD Biosciences,目录号:553080)
  10. APC结合的抗CD44抗体(克隆IM7)(BD Biosciences,目录号:559250)
  11. PE缀合的抗CD105抗体(克隆MJ7/18)(BD Biosciences,目录号:562759)
  12. FITC缀合的抗CD90抗体(克隆5E10)(BD Biosciences,目录号:555595)

设备

  1. 剪刀和镊子
  2. 注射器1cc,具有27规格×1/2针(BD,目录号:BD-309659和BD-305109)
  3. 培养皿100×20mm(BD,目录号:DL-353003)
  4. 50ml锥形管(Progene ,目录号:71-5000-B)
  5. 台式离心机
  6. 文化宫
  7. 血细胞计数器
  8. T-25烧瓶(BD,目录号:353108)
  9. 37℃,5%CO 2细胞培养箱
  10. 流式细胞仪(例如 BD LSRFortessa)

程序

  1. 在培养罩下,将来自一只6-8周龄小鼠的胫骨和股骨用不含FBS的DMEM在预先在37℃温热的培养皿中(见视频1)冲洗。

    视频1.鼠标骨髓收集
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  2. 用注射器和针头,吸出并弹出2-3次骨髓,以破坏它
  3. 转移到50ml锥形管中
  4. 在4℃下以430×g离心10分钟。
  5. 用补充有15%FBS和青霉素/链霉素的DMEM以10 6个细胞/ml的密度重悬沉淀物。
  6. 板在T25烧瓶中的1×10 7个细胞(10ml)
  7. 在37℃-5%CO 2下孵育7-10天而不更换培养基。
  8. 除去上清液,以保持只有粘附细胞,用5ml PBS洗涤烧瓶2次
  9. 胰蛋白酶细胞扩增。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。
  10. 7-10天后,通过流式细胞术评估生长的贴壁细胞的表型。
    1. 用异硫氰酸荧光素(FITC)标记的抗CD45(克隆30-F11)和用别藻蓝蛋白(APC)标记的抗CD31(克隆MEC133)染色细胞。这些抗体应稀释1/200。细胞应该对于这两种标记是阴性的,因为CD45是造血细胞标记物,CD31是内皮细胞标记物。
    2. 用1/200稀释的APC标记的抗CD44(克隆IM7),藻红蛋白(PE)标记的抗CD105(克隆MJ7/18)和FITC标记的抗CD90(克隆5E10) 。细胞应该对这三种标记是阳性的。

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)。 聚谷氨酸基序赋予IL-27羟基磷灰石和骨结合特性。 J Immunol 190(6):2931-2937。
  2. Tormo,A.J.,Beauséjour,C。和Gauchat,J.F。(2014)。 在体外绑定到分泌的骨基质 。 生物协议 4(4):e1030
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tormo, A. J., Rafei, M. and Gauchat, J. (2014). Isolation of Multipotent Stromal Cells from Mouse Bone Marrow. Bio-protocol 4(4): e1031. DOI: 10.21769/BioProtoc.1031.
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