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Isolation of Joint-infiltrating Cells
关节中免疫浸润细胞的分离   

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参见作者原研究论文

本实验方案简略版
Academic Radiology
May 2014

Abstract

Infiltration of leukocytes into joints is one of the main features of autoimmune inflammatory arthritis. Here, we describe the protocol for isolation of joint-infiltrating cells in mice. This protocol is useful to analyze cell surface antigens and intracellular cytokines by flow cytometry.

Keywords: Joint (关节), Arthritis (关节炎), Inflammation (炎症), Autoimmune disease (自身免疫性疾病)

Materials and Reagents

  1. 27-gauge needle (TERUMO, catalog number: NN-2719S )
  2. 1 ml syringe (TERUMO, catalog number: SS-01T )
  3. 6-well plate (Coring, Falcon®, catalog number: 353046 )
  4. 50 ml tube
  5. 70 μm cell strainer (Coring, Falcon®, catalog number: 352350 )
  6. 10 ml syringe (TERUMO, catalog number: SS-10SZ )
  7. Mice (adult > 6 weeks, any sex, any strain)
  8. Hyaluronidase (60 mg/ml stock) (Sigma-Aldrich, catalog number: H3506 )
  9. Collagenase type VIII (10 mg/ml stock) (Sigma-Aldrich, catalog number: C2139 )
  10. RPMI 1640 (Sigma-Aldrich, catalog number: R8758 )
  11. Fetal bovine serum, heat inactivated (Biowest, catalog number: S1820-500 )
  12. HANK’s solution “Nissui”② (NISSUI PHARMACEUTICAL, catalog number: 05906 )
  13. NaHCO3 (NAKARAI TESQUE, catalog number: 31212-25 )
  14. NaN3 (NAKARAI TESQUE, catalog number: 31208-82 )
  15. Digestion medium (see Recipes)
  16. FACS solution (see Recipes) 

Equipment

  1. Surgical scissors and tweezers
  2. Mini-shaker 3D (Biosan, catalog number: BS-010151-AAK )
  3. Refrigerated centrifuge

Procedure

  1. Euthanize mice. Sterilize mouse skin with 70% ethanol and peel off the skin of ankles using scissors and tweezers (Figure 1). Be careful not to leave any skin.


    Figure 1. Peel off the skin of ankles

  2. Cut out the leg at 0.7 cm above the ankle joint using scissors, and discard the finger portion (Figure 2).


    Figure 2. Cut out ankles

  3. Wash ankle portion in 2 ml RPMI 1640.
  4. Flush out bone marrow cells using a 27-gauge needle and 1 ml syringe filled with RPMI 1640 and discard the bone marrow cells.
  5. Put bone marrow-removed ankles in 4 ml of digestion medium on a 6-well plate (Figure 3).


    Figure 3. Put ankles in digestion medium

  6. Chop up ankles with scissors to 3-4 mm sized chunks (Figure 4).


    Figure 4. Chop up ankles

  7. Incubate ankles in a 6-well plate with gentle shaking by Mini-shaker for 1 h at 37 °C under 5% CO2.
  8. Place a cell strainer in a 50 ml tube. Then, transfer the medium with digested ankle pieces onto the cell strainer.
  9. Remove the plunger from a 10 ml syringe. Mash the residues of ankle pieces using the black rubber end of the plunger.
  10. Add 10 ml 10% FBS/RPMI 1640.
  11. Centrifuge at 1,500 rpm (400 x g) for 5 min at 4 °C.
  12. Discard the supernatant and resuspend the cell pellet in FACS solution.
  13. Count viable cells. Representative data are shown below (Figure 5).
  14. Perform FACS analysis. Representative data are shown below (Figure 6).

Representative data



Figure 5. Cell numbers in ankle joints. Total cell numbers (A) and T cell numbers (B) obtained by this protocol are shown. White diamonds represent cells from non-inflamed ankles in a WT mouse, and black diamonds represent cells from inflamed ankles in an Il1rn-/- mouse (Akitsu et al., 2015). Each symbol indicates number of joint cells without flushing (step 4) [Flush (-)], bone marrow-removed joint cells [(Flush (+)], and bone marrow cells (BM). Horizontal lines indicate medians.


Figure 6. An example of FACS analysis of joint-infiltrated cells from inflamed ankles. Flow cytometry of joint-infiltrating cells from Il1rn-/- mice (Akitsu et al., 2015). Numbers indicate percent cells in each gate. Data are representative of 10 independent experiments.

Notes

  1. Similar data were obtained by ten independent experiments using this protocol.
  2. If joints are arthritic with inflammation, joint cells from a mouse are enough to analyze with FACS. However, for normal joints, a pool of two to four mice is recommended. 
  3. From arthritic mice, 2-4 x 104 T cells are obtained from one mouse (without flushing step). Because bone marrow-derived cells are about 1/5 of the total T cells, flushing step can be skipped. On the other hand, total cell numbers without flushing step are approximately 4 x 106 cells/non-inflamed ankles, and 7-13 x 106 cells/inflamed ankles. Because 1/2-1/3 of them are derived from bone marrow, flushing step is necessary for analyzing non-T cell population. After the flushing step, total joint-infiltrated cell numbers are approximately 2 x 106 cells in non-inflamed ankles, and 5-7 x 106 cells in inflamed ankles.

Recipes

  1. Digestion medium
    160 μl hyaluronidase (final concentration: 2.4 mg/ml)
    400 μl collagenase VIII (final concentration: 1 mg/ml)
    4 ml of RPMI 1640 supplemented with 10% FBS
  2. FACS solution
    Dissolve 4.9 g HANK’s in 490 ml H2O
    Add 0.15 g NaHCO3 and 0.05 g NaN3, and dissolve completely
    Add 10 ml inactivated FBS (final 2%), then mix
    Store at 4 °C until use

Acknowledgments

This work was supported by a Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Akitsu, A., Ishigame, H., Kakuta, S., Chung, S. H., Ikeda, S., Shimizu, K., Kubo, S., Liu, Y., Umemura, M., Matsuzaki, G., Yoshikai, Y., Saijo, S. and Iwakura, Y. (2015). IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2(+)Vγ6(+)γδ T cells. Nat Commun 6: 7464.

简介

白细胞渗入关节是自身免疫性炎症性关节炎的主要特征之一。 在这里,我们描述的协议隔离的关节浸润细胞在小鼠。 该方案可用于通过流式细胞术分析细胞表面抗原和细胞内细胞因子。

关键字:关节, 关节炎, 炎症, 自身免疫性疾病

材料和试剂

  1. 27号针(TERUMO,目录号:NN-2719S)
  2. 1ml注射器(TERUMO,目录号:SS-01T)
  3. 6孔板(Coring,Falcon ,目录号:353046)
  4. 50ml管
  5. 70μm细胞过滤器(Coring,Falcon ,目录号:352350)
  6. 10ml注射器(TERUMO,目录号:SS-10SZ)
  7. 小鼠(成人> 6周,任何性别,任何应变)
  8. 透明质酸酶(60mg/ml储液)(Sigma-Aldrich,目录号:H3506)
  9. 胶原酶VIII型(10mg/ml储液)(Sigma-Aldrich,目录号:C2139)
  10. RPMI 1640(Sigma-Aldrich,目录号:R8758)
  11. 胎牛血清,热灭活(Biowest,目录号:S1820-500)
  12. HANK的解决方案"Nissui"②(NISSUI PHARMACEUTICAL,目录号:05906)
  13. NaHCO 3(NAKARAI TESQUE,目录号:31212-25)
  14. NaN 3(NAKARAI TESQUE,目录号:31208-82)
  15. 消化介质(参见配方)
  16. FACS解决方案(参见配方)

设备

  1. 外科剪刀和镊子
  2. Mini-shaker 3D(Biosan,目录号:BS-010151-AAK)
  3. 冷冻离心机

程序

  1. 安乐死小鼠。 用70%乙醇消毒小鼠皮肤,并用剪刀和镊子剥离踝关节的皮肤(图1)。 小心不要留下任何皮肤。


    图1.剥离踝关节的皮肤

  2. 使用剪刀剪断踝关节上方0.7厘米处的腿,并丢弃手指部分(图2)。


    图2.剪下脚踝

  3. 在2ml RPMI 1640中洗踝部分。
  4. 使用填充有RPMI 1640的27号针和1ml注射器冲洗骨髓细胞,并丢弃骨髓细胞。
  5. 将骨髓移除的踝关节置于6 ml板的4 ml消化培养基中(图3)。


    图3.将脚踝置于消化介质

  6. 用剪刀将脚踝剪成3-4毫米大小的块(图4)。


    图4.剪断踝关节

  7. 在6孔板中温育脚踝,在37℃,5%CO 2下,通过Mini-shaker轻轻摇动1小时。
  8. 将细胞过滤器置于50ml管中。 然后,将消化踝关节的介质转移到细胞滤网上
  9. 从10ml注射器中取出柱塞。 使用柱塞的黑色橡胶端将踝部残留物捣碎。
  10. 加入10ml 10%FBS/RPMI 1640.
  11. 在4℃下以1,500rpm(400×g)离心5分钟
  12. 弃去上清液,并在FACS溶液中重悬细胞沉淀
  13. 计数活细胞。 代表性数据如下所示(图5)。
  14. 执行FACS分析。 代表性数据如下所示(图6)。

代表数据



图5.踝关节中的细胞数。显示通过该方案获得的总细胞数(A)和T细胞数(B)。白色菱形代表来自WT小鼠的非发炎踝的细胞,黑色菱形代表来自Illrn -/- 小鼠中发炎踝的细胞(Akitsu, al 。,2015)。每个符号表示无冲洗的关节细胞数(步骤4)[冲洗( - )],去除骨髓的关节细胞[(冲洗(+)]和骨髓细胞>

图6.来自发炎踝关节浸润的细胞的FACS分析的实例来自II1rn -/- 的关节浸润细胞的流式细胞术。小鼠(Akitsu,et al。,2015)。数字表示每个门中的单元格百分比。数据代表10次独立实验。

笔记

  1. 通过使用该方案的十次独立实验获得类似的数据
  2. 如果关节炎是关节炎与炎症,来自小鼠的关节细胞足以用FACS分析。但是,对于正常关节,推荐使用两到四只小鼠的水池。
  3. 从关节炎小鼠,从一只小鼠获得2-4×10 4个T细胞(不冲洗步骤)。因为骨髓来源的细胞是总T细胞的约1/5,所以可以跳过冲洗步骤。另一方面,没有冲洗步骤的总细胞数为约4×10 6个细胞/非发炎踝和7-13×10 6个细胞/发炎踝。因为1/2-1/3来源于骨髓,所以需要冲洗步骤来分析非T细胞群体。在冲洗步骤后,全部关节浸润的细胞数在非发炎踝关节中为约2×10 6个细胞,在发炎的踝关节中为约5-7×10 6个细胞。

食谱

  1. 消化介质
    160μl透明质酸酶(终浓度:2.4mg/ml) 400μl胶原酶VIII(终浓度:1mg/ml) 4ml补充有10%FBS的RPMI 1640
  2. FACS解决方案
    将4.9g HANK溶解在490ml H 2 O中 加入0.15g NaHCO 3和0.05g NaN 3,并完全溶解
    加入10ml灭活的FBS(最终2%),然后混合
    在4°C储存,直到使用

致谢

这项工作得到了日本教育,文化,体育,科学和技术部的助学金的支持。

参考文献

  1. Akitsu,A.,Ishigame,H.,Kakuta,S.,Chung,SH,Ikeda,S.,Shimizu,K.,Kubo,S.,Liu,Y.,Umemura,M.,Matsuzaki,G.,Yoshikai ,Y.,Saijo,S。和Iwakura,Y。(2015)。  Nat Commun 6:7464。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Akitsu, A. and Iwakura, Y. (2016). Isolation of Joint-infiltrating Cells. Bio-protocol 6(17): e1911. DOI: 10.21769/BioProtoc.1911.
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