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Bone Marrow Mesenchymal Stem Cells Adhesion Assay
骨髓间充质干细胞粘附性测定   

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Abstract

Mesenchymal stem cells (MSCs) are widespread in adult organisms and involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. As cell adhesion play important roles in cell interactions and signaling, to thoroughly evaluate the adhesion ability of MSCs is of vital importance to clarify the mechanism of self-renewal, proliferation, activation and migration of MSCs in different microenvironments. Based on the method by Siler et al., 2000, we revised the protocol in order to provide details on how to evaluate the adhesion ability of MSCs from bone marrow (BMSCs) on extracellular matrix (ECM) protein laminins. The current protocol can also be easily translated to MSCs with other treatments or ECMs such as collagens, fibronectin, etc.

Keywords: Mesenchymal stem cells(间充质干细胞), Extracellular matrix(细胞外基质), Adhesion(粘附), Stem cells(干细胞), Laminin(层粘连蛋白)

Materials and Reagents

  1. 96 well culture plate (Sigma-Aldrich, Corning® Costar®, catalog number: CLS3595 )
  2. Human bone marrow derived MSCs, isolated and cultured as previously described (Kern et al., 2006)
    Note: Cells in the third cell passage were used.
  3. Laminin 511&521 (Biolamina, catalog number: LN511 ; LN521 )
  4. DMEM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11965092 )
  5. Fetal bovine serum (FBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10099141 )
  6. Trypsin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25200-056 )
  7. Soybean trypsin inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17075-029 )
  8. BSA (Sigma-Aldrich, catalog number: V900933 )
  9. Blocking buffer (0.5% BSA in PBS without Ca2+ and Mg2+)
  10. Wash buffer (0.1% BSA in PBS without Ca2+ and Mg2+)
  11. 2% SDS (Sigma-Aldrich, catalog number: 71729 )
  12. 4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  13. NaCl (Sigma-Aldrich, catalog number: S7653 )
  14. KCl (Sigma-Aldrich, catalog number: 746436 )
  15. Na2HPO4 (Sigma-Aldrich, catalog number: 795410 )
  16. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
  17. CaCl2·2H2O (Sigma-Aldrich, catalog number: 223506 )
  18. MgCl2·6H2O (Sigma-Aldrich, catalog number: M9272 )
  19. HCl (Sinopharm chemical reagent Being Co.ltd, catalog number: 10011008 )
  20. Crystal violet powder (Sigma-Aldrich, catalog number: C6158 )
  21. Ethanol (Sinopharm chemical reagent Being Co.ltd, catalog number: 10009159 )
  22. PBS with/without Ca2+ and Mg2+ (see Recipes)
    Note: Commercial DPBS with/without calcium and magnesium can also be used.
  23. 0.1% crystal violet staining solution (see Recipes)

Equipment

  1. Small shaker for microtiter plate (IKA, model: MS3 Digital )
  2. Scanner (UMAX, model: POWERLOOK 2100XL )
  3. Series II 3110 Water-Jacketed CO2 chamber (Thermo Fisher Scientific, FormaTM, catalog number: 3111 )
  4. Microscope (Nikon, model: ECLIPSE TE2000-S )
  5. Microplate reader (ThermoFisher Scientific, model: Multiscan MK3 ) or spectrometer with 550 nm wavelength available.

Procedure

  1. Coat plate with laminins
    1. Slowly thaw recombinant laminins at 2 °C to 8 °C before use.
    2. Dilute the thawed laminin stock solution with 1x DPBS (Ca2+/Mg2+) to 10 μg/ml, Add 60 μl diluted laminin solution to each well of 96 well culture plate (The final coating concentration is 2 μg/cm2). Make sure the laminin solution is spread evenly across the surface. Leave some wells uncoated as negative control.
    3. Incubate at 2 °C to 8 °C overnight.
    4. Wash with wash buffer (200 μl/well) for 2 times.
    5. Block plates with blocking buffer (200 μl/well) at 37 °C in CO2 chamber for 60 min.
    6. Wash with wash buffer (200 μl/well) for 2 times.
    Note: All the wash procedures do not need some time incubation, the followings are the same.
  2. Prepare and seed cells
    1. Taking a 10 cm culture dish as an example, wash the cells with 2 ml PBS.
    2. Detach the cells with 2 ml 0.25% Trypsin-EDTA, neutralize trypsin with 2 ml 0.25 mg/ml soybean trypsin Inhibitor.
    3. Mix the cells by pipetting up and down using a 1 ml pipette, then transfer them to a 15 ml sterile tube and centrifuge at 300 x g for 5 min.
    4. Resuspend the cells in 2 ml DMEM with 0.5% FBS.
    5. Count the cells and dilute to a final concentration of 2 x 105/ml, add 100 μl (2 x 104 cells) to each well. Set up at least triplicate wells for each condition.
      Note: Cell number vary depending on the coated ECM, for laminins, 2 x 104 is appropriate. However, 4 x 105 cells may be required for non-coated wells.
    6. Incubate at 37 °C (in a CO2 chamber) for 20 min.
      Note: Adhesion time depends on the coated ECM. It is useful to observe the adherent condition under microscope at different time points, choosing between 5 min and 30 min (5, 10, 15, 20, 30), even do a time course first. In our system, cells adhere to the laminin-coated wells within at least 10 min, while for non-coated wells, it may take longer time.
  3. Stop the assay
    1. Discard the seeding medium carefully without scratching the bottom of wells.
    2. Rinse cells once with 200 μl PBS per well.
    3. Shake the plate at 2,000 rpm for 15 sec.
    4. Discard the PBS and wash with 200 μl wash buffer per well for 3 times.
    5. Fix with 200 μl 4% paraformaldehyde per well. Incubate at room temperature for 10-15 min.
    6. Wash with 200 μl PBS per well.
  4. Staining and analysis
    1. Add 100 μl 0.1% crystal violet to each well and incubate for 10 min at room temperature.
    2. Remove the crystal violet and wash with ddH2O (200 μl/well) for 3 times.
    3. Turn the plates upside down on the absorbent papers. Let the plates dry up completely.
    4. The plates can be scanned by a scanner firstly.
    5. Add 100 μl 2% SDS to each well and incubate for 10 min with gently shaking at room temperature.
    6. Read the absorbance within 5 min on a microplate reader/spectrometer at a wavelength of 550 nm.

Representative data



Figure 1. Laminin 511 promotes the adhesion of bone marrow mesenchymal stem cells (BMSCs). A. The scanned image of crystal violet staining of BMSCs adhered to laminin 511 and BSA coated wells; B. The O.D. values from the dissolved crystals are shown for (A), the data are presented as the mean ± SEM from three replicate wells; ***, P < 0.001.

Recipes

  1. PBS with/without Ca2+ and Mg2+ (Reference 1)
    1. Dissolve the following in 800 ml distilled H2O
       8 g NaCl
       0.2 g KCl
       1.44 g Na2HPO4
       0.24 g KH2PO4
       For PBS with Ca2+ and Mg2+, supplement with the following
       0.133 g CaCl2·2H2O
       0.10 g MgCl2·6H2O
    2. Adjust pH to 7.4 with HCl
    3. Adjust volume to 1 L with additional distilled H2O
    4. Sterilize by autoclaving
  2. 0.1% crystal violet staining solution
    To make up 50 ml crystal violet solution, dissolve 50 mg crystal violet powder in (5 ml ethanol/45 ml water).

References

  1. Phosphate-buffered saline (PBS). (2006) Cold Spring Harb Protoc: doi:10.1101/pdb.rec8247.
  2. Kern, S., Eichler, H., Stoeve, J., Kluter, H. and Bieback, K. (2006). Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24(5): 1294-1301.
  3. Siler, U., Seiffert, M., Puch, S., Richards, A., Torok-Storb, B., Muller, C. A., Sorokin, L. and Klein, G. (2000). Characterization and functional analysis of laminin isoforms in human bone marrow. Blood 96(13): 4194-4203.

简介

间充质干细胞(MSC)在成年生物体中广泛存在并参与组织维持和修复以及造血作用和免疫反应的调节。 由于细胞粘附在细胞相互作用和信号转导中发挥重要作用,彻底评估MSC的粘附能力对于阐明MSC在不同微环境中的自我更新,增殖,活化和迁移的机制至关重要。 基于Siler等人的方法(2000),我们修订方案以提供关于如何评价来自骨髓(BMSCs)的MSC对细胞外基质(ECM)蛋白的粘附能力的细节 层粘连蛋白。 目前的方案也可以很容易地翻译成MSC与其他治疗或ECM,如胶原,纤连蛋白,等。

关键字:间充质干细胞, 细胞外基质, 粘附, 干细胞, 层粘连蛋白

材料和试剂

  1. 96孔培养板(Sigma-Aldrich,Corning ,目录号:CLS3595)。
  2. 人骨髓来源的MSC,如先前所述分离和培养(Kern等人,2006)
    注意:使用第三次传代的细胞。
  3. 层粘连蛋白511和521(Biolamina,目录号:LN511; LN521)
  4. DMEM(Thermo Fisher Scienfitic,Invitrogen TM ,目录号:11965092)
  5. 胎牛血清(FBS)(Thermo Fisher Scienfitic,Invitrogen TM ,目录号:10099141)
  6. 胰蛋白酶(Thermo Fisher Scienfitic,Invitrogen TM ,目录号:25200-056)
  7. 大豆胰蛋白酶抑制剂(Thermo Fisher Scienfitic,Invitrogen TM,目录号:17075-029)
  8. BSA(Sigma-Aldrich,目录号:V900933)
  9. 封闭缓冲液(不含Ca 2+和Mg 2+的PBS中的0.5%BSA)
  10. 洗涤缓冲液(不含Ca 2+和Mg 2+的PBS中的0.1%BSA)
  11. 2%SDS(Sigma-Aldrich,目录号:71729)
  12. 4%多聚甲醛(Sigma-Aldrich,目录号:158127)
  13. NaCl(Sigma-Aldrich,目录号:S7653)
  14. KCl(Sigma-Aldrich,目录号:746436)
  15. Na 2 HPO 4(Sigma-Aldrich,目录号:795410)。
  16. KH sub 2 PO 4(Sigma-Aldrich,目录号:P0662)
  17. CaCl 2·2H 2 O(Sigma-Aldrich,目录号:223506)
  18. MgCl 2·6H 2 O(Sigma-Aldrich,目录号:M9272)
  19. HCl(Sinopharm chemical reagent Being Co.ltd,目录号:10011008)
  20. 结晶紫色粉末(Sigma-Aldrich,目录号:C6158)
  21. 乙醇(Sinopharm chemical reagent Being Co.ltd,目录号:10009159)
  22. 具有/不具有Ca 2+ 2 + 和Mg 2+ 2 + 的PBS PBS(参见配方)
    注意:也可以使用含/不含钙和镁的商品DPBS。
  23. 0.1%结晶紫染色溶液(见配方)

设备

  1. 用于微量滴定板(IKA,型号:MS3Digital)的小振荡器
  2. 扫描仪(UMAX,型号:POWERLOOK 2100XL)
  3. 系列II 3110水夹套CO 2室(Thermo Fisher Scientific,Forma TM ,目录号:3111)
  4. 显微镜(Nikon,型号:ECLIPSE TE2000-S)
  5. 微孔板读数器(ThermoFisher Scientific,型号:Multiscan MK3)或具有550nm波长的光谱仪

程序

  1. 用层粘连蛋白包被板
    1. 在使用前,将重组体层粘连蛋白在2°C至8°C缓慢解冻
    2. 用1×DPBS(Ca 2+ 2 + /Mg 2 + )稀释解冻的层粘连蛋白储备溶液至10μg/ml,向96孔的每个孔中加入60μl稀释的层粘连蛋白溶液 培养板(最终涂层浓度为2μg/cm 2)。 确保层粘连蛋白溶液均匀地铺展在整个表面上。 将一些孔未涂布为阴性对照。
    3. 在2°C至8°C孵育过夜
    4. 用洗涤缓冲液(200μl/孔)洗涤2次。
    5. 在37℃下在CO 2室中用封闭缓冲液(200μl/孔)封闭板60分钟。
    6. 用洗涤缓冲液(200μl/孔)洗涤2次。
    注意:所有洗涤程序不需要一些时间孵育,以下是相同的。
  2. 准备和种子细胞
    1. 以10cm培养皿为例,用2ml PBS洗涤细胞
    2. 用2ml 0.25%胰蛋白酶-EDTA分离细胞,用2ml 0.25mg/ml大豆胰蛋白酶抑制剂中和胰蛋白酶。
    3. 通过用1ml移液管上下吹吸混合细胞,然后将其转移到15ml无菌管中,并以300xg离心5分钟。
    4. 将细胞重悬在含有0.5%FBS的2ml DMEM中
    5. 计数细胞并稀释至2×10 5个/ml的终浓度,向每个孔中加入100μl(2×10 4个细胞)。对每种条件设置至少三次重复的孔。
      注意:细胞数量根据包被的ECM而不同,对于层粘连蛋白,2×10 4 是适当的。但是,非涂层孔可能需要4 x 10 5 个细胞。
    6. 在37℃(在CO 2室中)孵育20分钟。
      注意:粘附时间取决于涂层的ECM。在不同的时间点观察显微镜下的粘附条件是有用的,在5分钟至30分钟(5,10,15,20,30)之间选择,甚至首先进行时间进程。在我们的系统中,细胞在至少10分钟内粘附于层粘连蛋白包被的孔,而对于未包被的孔,可能需要更长的时间。
  3. 停止测试
    1. 小心弃去播种介质,不要划伤井底。
    2. 每孔用200μlPBS冲洗细胞一次。
    3. 在2000 rpm下摇动平板15秒。
    4. 弃去PBS,并用200μl洗涤缓冲液每孔洗涤3次。
    5. 固定与200微升4%多聚甲醛每孔。 在室温下孵育10-15分钟。
    6. 每孔用200μlPBS洗涤。
  4. 染色和分析
    1. 向每个孔中加入100μl0.1%结晶紫,并在室温下孵育10分钟。
    2. 除去结晶紫,并用ddH 2 O(200μl/孔)洗涤3次。
    3. 在吸水纸上翻转平板。 让板块完全干燥。
    4. 可以首先用扫描仪扫描板
    5. 向每个孔中加入100μl2%SDS,并在室温下轻轻摇动孵育10分钟
    6. 在微板读数器/光谱仪上在550nm的波长下在5分钟内读取吸光度。

代表数据



图1.层粘连蛋白511促进骨髓间充质干细胞(BMSCs)的粘附。 A.BMSC的结晶紫染色的扫描图像粘附在层粘连蛋白511和BSA包被的孔上; B. O.D. (A)显示来自溶解晶体的值,数据表示为来自三个重复孔的平均值±SEM; ***, P 0.001。

食谱

  1. 具有/不具有Ca 2+和Mg 2+的PBS的PBS(参考文献1)
    1. 将以下物质溶于800ml蒸馏H 2 O中   8克NaCl
        0.2 g KCl
        1.44g Na sub 2 HPO 4
        0.24 g KH <2> PO 4
       对于含Ca 2 + 和Mg 2 + 的PBS,请补充以下
        0.133克CaCl 2 2·2H 2 O
      0.10g MgCl 2·6H 2 O·dm /
    2. 用HCl
      调节pH至7.4
    3. 用额外的蒸馏H 2 O 2调节体积至1L
    4. 高压灭菌
      灭菌
  2. 0.1%结晶紫染色溶液
    为了补足50毫升结晶紫溶液,将50毫克结晶紫粉末溶解在(5毫升乙醇/45毫升水)中

参考文献

  1. 磷酸盐缓冲盐水(PBS)。  (2006) Cold Spring Harb Protoc :doi:10.1101/pdb.rec8247。
  2. Kern,S.,Eichler,H.,Stoeve,J.,Kluter,H。和Bieback,K。(2006)。  来自骨髓,脐带血或脂肪组织的间充质干细胞的比较分析干细胞 24(5 ):1294-1301。
  3. Siler,U.,Seiffert,M.,Puch,S.,Richards,A.,Torok-Storb,B.,Muller,CA,Sorokin,L。和Klein,G。(2000) "ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/11110691"target ="_ blank">人骨髓中层粘连蛋白亚型的表征和功能分析血液 96(13):4194-4203。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yang, Z. and Xiao, R. (2016). Bone Marrow Mesenchymal Stem Cells Adhesion Assay. Bio-protocol 6(15): e1895. DOI: 10.21769/BioProtoc.1895.
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