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Bone Marrow Derived Eosinophil Cultures
骨髓源嗜酸性细胞培养   

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Abstract

Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection. Mouse bone marrow derived progenitor cells can be differentiated into eosinophils following IL-5 exposure. These bone marrow derived eosinophils are fully differentiated at the end of a 14 day culture based on morphology and expression of molecular markers.

Materials and Reagents

  1. Mice
  2. Histopaque 1083 (Sigma-Aldrich, catalog number: 10831 -100ml)
  3. IMDM with Glutamax-I (Life Technologies, InvitrogenTM, catalog number: 31980-097 )
  4. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  5. Penicillin/Streptomycin (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
  6. Stem Cell Factor (Pepro tech, catalog number: 250-03 )
  7. FLT-3 Ligand (Pepro tech, catalog number: 250-31L )
  8. Recombinant mouse IL-5 (Pepro tech, catalog number: 215-15 )
  9. PBS (Life Technologies, InvitrogenTM, catalog number: 14200-166 )
  10. Diff-Quick Stain Kit (Thermo Fisher Scientific, catalog numbers: 23-122-929 , 23-122-952 , and 23-122-937 )
  11. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 -100ml)
  12. CCR3 (R&D systems, Catalog number: FAB729F )
  13. Siglec-F (BD, PharmingenTM, Catalog number: 552126 )
  14. IMDM cell culture media (see Recipes)

Equipment

  1. 6-well tissue culture plate
  2. Dissection tools: scalpel, scissors
  3. 1 ml syringe
  4. 15 ml sterile centrifuge tube
  5. Centrifuge
  6. 37 °C, 5% CO2 cell culture incubator
  7. Microscope
  8. Hemocytometer

Procedure

  1. Collect femur/tibia from 6-8 mice.
    Note: Mice between 6-8 weeks old will give the highest yield of low-density bone marrow fraction.
  2. Flush the bone marrow from femur/tibia with PBS, pipet up and down to break up the bone marrow into single cell suspension.
  3. Spin down the cells at 300 x g 8 min at 4 °C.
  4. Resuspend cells in 15 ml of RBC lysis buffer, incubate for 2 min at 37 °C.
    Note: If less than 6-8 mice were used, there is no need to adjust the volume of RBC lysis buffer. If more than 6-8 mice were used, we recommend scaling up the RBC lysis buffer accordingly and also scaling up the amount of PBS used to neutralize the RBC lysis buffer in step 5.
  5. Add 30 ml of PBS to neutralize the RBC lysis buffer.
  6. Spin down the cells at 300 x g 8 min at 4 °C.
  7. Resuspend the cells in 16 ml of PBS.
  8. Add 4 ml of histopaque 1083 to each of 15 ml centrifuge tube.
  9. Carefully layer 4 ml of cells from step 7 to 4 ml of histopage 1083 prepared in step 8. Must use very slow speed to ensure that the layers are not disturbed. At the end of this step, there should be two layers: bottom clear layer contains histopage 1083, and top cloudy layer contaning cells.
  10. Centrifuge at 400 x g for exactly 30 min at room temperature. The brake on the centrifuge needs to be in the off position.
  11. Collect the low density fraction with a 1 ml micropipette. The low density fraction is at the interface between upper and lower layers. The approximate volume of this fraction is 0.5 ml to 1 ml.
    Note: The low-density fraction is cloudy due to the low-density bone marrow cells at this fraction. The upper layer immediately above it is clear. The lower layer immediately below it should be significantly less cloudy compared to the low-density fraction.
  12. Wash the cells with 30 ml of PBS and spin down at 300 x g 8 min.
  13. Resusped the cells in IMDM with Glutamax-1 with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin supplemented with 100 ng/ml stem cell factor and 100 ng/ml FLT-3 ligand at a concentration of 1 x 106/ml.
  14. Plate 3 x 106 cells per well in 6 well plates (3 ml).
  15. Change culture media on day 2 by aspirating 1.5 ml of media from each well and adding 1.5 ml of new media (see Recipes).
    Note: Be careful not to aspirate the cells as the cells are settled to the bottom of the plate but are not attached.
  16. On day 4 , collect all the cells, spin down the cells at 300 x g 8 min at 4 °C, wash once with 30 ml of PBS, then resuspend the cells at a concentration of 1 x 106/ml in IMDM with Glutamax-1 with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin supplemented with 10 ng/ml of IL-5.
  17. Change the culture culture media every other day. Cells should be counted and concentration adjusted to 1 x 106/ml during each media change.
  18. At day 14, collect mature eosinophils for further studies. Eosinophil maturity can be assessed by FACS staining for CCR3 and Siglec-F and/or Diff-Quik staining of cytospin preparations following the manufacturer’s protocol (Figure 1).


    Figure 1. Morphology of mature bone marrow derived eosinophils. Morphology determined by Diff-Quik staining following the manufacturer’s protocol.

Recipes

  1. IMDM cell culture media
    IMDM with Glutamax-1
    10% FBS
    100 U/ml penicillin
    100 μg/ml streptomycin supplemented with 100 ng/ml stem cell factor
    100 ng/ml FLT-3 ligand
    Cellular concentration of 1 x 106/ml

Acknowledgments

This protocol has been adapted from Lu et al. (2013).

References

  1. Lu, T. X., Lim, E. J., Besse, J. A., Itskovich, S., Plassard, A. J., Fulkerson, P. C., Aronow, B. J. and Rothenberg, M. E. (2013). MiR-223 deficiency increases eosinophil progenitor proliferation. J Immunol 190(4):1576-1582.

简介

嗜酸性粒细胞是涉及多种疾病的发病机理的多功能效应细胞,包括哮喘,嗜酸性粒细胞胃肠道疾病和蠕虫感染。 小鼠骨髓来源的祖细胞可以在IL-5暴露后分化成嗜酸性粒细胞。 这些骨髓衍生的嗜酸性粒细胞在14天培养结束时基于分子标志物的形态学和表达完全分化。

材料和试剂

  1. 小鼠
  2. Histopaque 1083(Sigma-Aldrich,目录号:10831-100ml)
  3. IMDM与Glutamax-1(Life Technologies,Invitrogen TM ,目录号:31980-097)
  4. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150)
  5. 青霉素/链霉素(Life Technologies,InvitrogenTM,目录号:15140-122)
  6. 干细胞因子(Pepro tech,目录号:250-03)
  7. FLT-3配体(Pepro tech,目录号:250-31L)
  8. 重组小鼠IL-5(Pepro tech,目录号:215-15)
  9. PBS(Life Technologies,Invitrogen TM ,目录号:14200-166)
  10. Diff-Quick染色试剂盒(Thermo Fisher Scientific,目录号:23-122-929,23-122-952和23-122-937)
  11. RBC裂解缓冲液(Sigma-Aldrich,目录号:R7757-100ml)
  12. CCR3(R& D systems,目录号:FAB729F)
  13. Siglec-F(BD,Pharmingen TM ,目录号:552126)
  14. IMDM细胞培养基(参见配方)

设备

  1. 6孔组织培养板
  2. 解剖工具:解剖刀,剪刀
  3. 1 ml注射器
  4. 15 ml无菌离心管
  5. 离心机
  6. 37℃,5%CO 2细胞培养箱中培养
  7. 显微镜
  8. 血细胞计数器

程序

  1. 从6-8只小鼠收集股骨/胫骨。
    注意:6-8周龄的小鼠将产生最低的低密度骨髓组分。
  2. 用PBS冲洗来自股骨/胫骨的骨髓,用移液管上下移动,将骨髓破碎成单细胞悬浮液。
  3. 在4℃下以300×g离心细胞8分钟。
  4. 重悬细胞在15毫升RBC裂解缓冲液,孵育2分钟,在37℃。
    注意:如果使用少于6-8只小鼠,则不需要调整RBC裂解缓冲液的体积。 如果使用超过6-8只小鼠,我们建议相应地按比例放大RBC裂解缓冲液,并在步骤5中放大用于中和RBC裂解缓冲液的PBS量。
  5. 加入30ml PBS以中和RBC裂解缓冲液。
  6. 在4℃下,在300×g下旋转细胞8分钟。
  7. 将细胞重悬在16ml PBS中
  8. 在15ml离心管中各加入4ml histopaque 1083
  9. 小心地将4ml来自步骤7的细胞层压至4ml在步骤8中制备的梯度1083。必须使用非常慢的速度以确保层不被扰乱。 在该步骤结束时,应该有两个层:底部透明层包含页码1083和顶部混浊层接纳单元。
  10. 在室温下以400×g离心30分钟。 离心机上的制动器需要处于关闭位置。
  11. 收集低密度部分与1毫升微量移液管。低密度部分在上层和下层之间的界面处。该级分的近似体积为0.5ml至1ml。
    注意:低密度部分由于该部分的低密度骨髓细胞而混浊。其正上方的上层是透明的。与低密度部分相比,其正下方的下层应显着较少混浊。
  12. 用30ml PBS洗涤细胞并在300×g下旋转8分钟
  13. 将IMDM中的细胞用含有10%FBS,100U/ml青霉素和100μg/ml链霉素的Glutamax-1补充有100ng/ml干细胞因子和100ng/ml FLT-3配体,浓度为1x 10 6 /ml
  14. 板在6孔板(3ml)中每孔3×10 6个细胞/孔。
  15. 在第2天更换培养基,从每个孔吸取1.5毫升培养基,加入1.5毫升新培养基(见Recipes)。
    注意:小心不要吸出细胞,因为细胞被固定在培养板的底部,但没有附着。
  16. 在第4天,收集所有细胞,在4℃下以300×g离心8分钟,用30ml PBS洗涤一次,然后以1×10 6个细胞/在含有Glutamax-1和10%FBS,100U/ml青霉素和100μg/ml补充有10ng/ml IL-5的链霉素的IMDM中培养6×10 6/ml。
  17. 每隔一天更换培养基。在每次培养基更换期间,应该计数细胞并将浓度调节至1×10 6/ml/ml。
  18. 在第14天,收集成熟嗜酸性粒细胞进行进一步研究。根据制造商的方案,可以通过CCR3和Siglec-F的FACS染色和/或细胞离心涂片制备物的Diff-Quik染色来评估嗜酸性粒细胞成熟度(图1)。


    图1.成熟骨髓来源的嗜酸性粒细胞的形态学。根据制造商的方案通过Diff-Quik染色确定形态。

食谱

  1. IMDM细胞培养基
    与Glutamax-1的IMDM
    10%FBS
    100 U/ml青霉素
    100μg/ml链霉素,补充有100ng/ml干细胞因子 100ng/ml FLT-3配体 细胞浓度为1×10 6/ml/ml

致谢

该协议已经从Lu等人修改(2013)。

参考文献

  1. Lu,T.X.,Lim,E.J.,Besse,J.A.,Itskovich,S.,Plassard,A.J.,Fulkerson,P.C.,Aronow,B.J.and Rothenberg,M.E。 MiR-223缺陷增加嗜酸性粒细胞祖细胞增殖。 J Immunol 190(4):1576-1582。
  • English
  • 中文翻译
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lu, T. X. and Rothenberg, M. E. (2014). Bone Marrow Derived Eosinophil Cultures. Bio-protocol 4(12): e1161. DOI: 10.21769/BioProtoc.1161.
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