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Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells
小鼠小肠隐窝细胞的分离和流式细胞术分析   

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Abstract

The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).

Keywords: Flow cytometry(流式细胞术), Colon(结肠), Intestinal(肠), Crypt cell(腺窝细胞), Mouse(小鼠)

Materials and Reagents

  1. Falcon 50 ml conical tubes (VWR International, catalog number: 21008-240 )
  2. Falcon 15 ml conical tubes (VWR International, catalog number: 21008-216 )
  3. 10 cm Petri dishes (Corning, Falcon®, catalog number: 353046 )
  4. Gauze, mesh size approximately 100 μm (Tegosa, catalog number: 10018 )
  5. Cell strainers (70 μm) (Corning, Falcon®, catalog number: 352350 )
  6. Cell strainers (40 μm) (Corning, Falcon®, catalog number: 352340 )
  7. 25G needle (Premier Healthcare & Hygiene, BD Microlance, catalog number: 300600 )
  8. 5 ml syringe (BD, catalog number: 309649)
  9. 5 ml round-bottom flow cytometry tube (BD Biosciences, catalog number: 352063 )
  10. 8- to 10-week old mice (Mus musculus) (male or female) (we used the C57BL/6 strain)
  11. Hank’s balanced salt solution medium (HBSS) (Life Technologies, Gibco®, catalog number: 2420-091 )
  12. Penicillin-Streptomycin Solution 100x (Biowest, catalog number: L0022 )
  13. Ethylenediaminetetraacetic acid (EDTA) (AppliChem GmbH, Panreac, catalog number: 13102/ 131026 )
  14. Fetal bovine serum (FBS) (Life Technologies, catalog number: GCS0103-500 )
  15. APC-conjugated anti-CD45 antibody (Beckman Coulter, catalog number: 732158 )
  16. FITC-conjugated anti-CD4 antibody (Beckman Coulter, catalog number: 731999 )
  17. PE-conjugated anti-Ly6G antibody (Beckman Coulter, catalog number: 732487 )
  18. Biotinylated anti-F480 antibody (eBioscience, catalog number: 13-4801 )
  19. PeyC7-conjugated anti-CD8 antibody (Biolegend, catalog number: 100721 )
  20. ECD-conjugated streptavidin (Beckman Coulter, catalog number: IM3326 )
  21. Dispase II (Life Technologies, catalog number: 17105-041 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17105-041”.
  22. Phosphate-buffered saline (PBS) (see Recipes)
  23. Disaggregation medium (see Recipes)

Equipment

  1. Dissection equipment (forceps & scissors)
  2. Scalpel or razor blade
  3. 37 °C chamber (Thermo Fisher Scientific, HeraeusTM)
  4. Refrigerated centrifuge (Thermo Fisher Scientific, HeraeusTM, model: MegafugeTM 2.012 )
  5. Neubauer chamber cell counter
  6. Orbital shaking platform (Kühner AG, Lab-Shaker)
  7. CO2 chamber
  8. Cytomics fc500 flow cytometer (Beckman Coulter)
  9. Microscope (ZEISS, model: Axiovert 40CFL )

Procedure

  1. Isolation of intestinal crypt cells
    1. Sacrifice mice in a CO2 chamber by trained personnel. Perform the inhalation of CO2 from a pressurized tank in a chamber constructed of clear material (e.g., Plexiglas) (a standard size mouse cage may contain no more than 5 mice) followed by cervical dislocation. According to the Institutional Animal Care and Use Committee, CO2 must be delivered in a controlled manner, at a low-flow rate of 10-30% volume displacement per minute.
    2. After swabbing the abdomen with alcohol, make a ventral incision. Use forceps and scissors to carefully remove the entire colon (Figure 1). Cut from the cecum to the anus (between 8-10 cm depending on the mouse size), and place it in a 10 cm Petri dish with 15 ml cold PBS to cover the colon.
    3. Hold the distal part of the colon with the forceps and flush to remove feces with 5-10 ml cold sterile PBS using a 25 G needle attached to a 5 ml syringe. After flushing out feces, place the cleaned colon in a new Petri dish with 10 ml cold PBS.
    4. Make a longitudinal incision in the colon and rinse again with cold PBS to remove remaining feces.
    5. Place the open colon in cold HBSS medium on ice until all colons for the experiment have been extracted as in steps A1-4. For good cell yield, we use three mice per test condition.
    6. Incubate all colons in 5 ml HBSS containing 100 U/ml penicillin and 0.1 mg/ml streptomycin (15 min, room temperature).
    7. Mince the colon tissue into small pieces of approx. 2 mm x 2 mm on a Petri dish, with the help of a scalpel. Place the small pieces in a 50 ml Falcon tube and incubate in 5 ml of 8 mM EDTA in HBSS (160 μl 0.5 M EDTA in 10 ml HBSS; 15 min, 37 °C).
    8. Remove the medium and add 5 ml fresh cold HBSS to the colon pieces in the bottom of the falcon tube.
    9. Dissociate crypt epithelial cells by vigorous hand shaking to obtain a supernatant enriched in crypts. Decant supernatant from tissue debris into a new Falcon 50 ml tube. Add fresh cold HBSS to the sample and repeat the shaking step. Repeat this procedure 3-5 times to increase crypt enrichment.
    10. To confirm crypt separation, visualize a 20 μl supernatant sample under the microscope (Figure 2).
    11. Centrifuge supernatants (200 x g, 15 min, 4 °C).
    12. Remove supernatant. To the pellets, add 5 ml of disaggregation medium, transfer them to a 15 ml Falcon tube and incubate (30 min, 37 °C at 2,500 rpm with orbital shaking) to obtain single-cell suspensions.
    13. Add 250 μl FBS to the medium (5% final concentration) after disaggregation to terminate the dispase reaction.
    14. Filter cells sequentially through 100 μm gauze, then 70 and 40 μm cell strainers.
    15. Collect the cells by centrifugation (150 x g, 10 min, 4 °C), resuspend in 1.5 ml staining buffer (2% FBS in PBS) and count live cells in a cell counter. The expected cell yield from three mice is approx. 2 x 107 cells.
    16. Stain 1 x 106 intestinal crypt cells with fluorescence-labeled antibodies to the cell markers of interest using standard FACS analysis procedures, and analyze on a flow cytometer.

  2. Example of flow cytometry analysis
    1. Incubate 1 x 106 intestinal crypt cells in 200 μl staining buffer (2% FBS in PBS) with fluorescently labeled anti-CD45, -CD4, -F4/80, -CD8 and -Ly6G antibodies (20 min, 4 °C, protected from light). Dilute the antibodies in PBS. 4-CD45-APC diluted 1/100; CD4-FITC diluted 1/100; F4/80-Biotina diluted 1/200; CD8-PeyC7 diluted 1/200 and Ly6G-PE diluted 1/100.
    2. Wash twice with 150 μl staining buffer, centrifuge (150 x g, 5 min, 4 °C) and discard supernatant.
    3. Incubate cells with streptavidin diluted 1:50 (20 min, 4 °C, protected from light).
    4. Repeat washing step B2.
    5. Resuspend cells in 200 μl staining buffer and transfer into round-bottom flow cytometry tubes with 220 μl staining buffer.
    6. Analyze staining with the Kaluza program, gating all positive cells for each antibody in CD45+ cells (Figure 3).

Representative data


Figure 1. Sequential images of colon isolation. Pictures were acquired sequentially as described in the text.


Figure 2. Light microscope images of mouse colon crypts. 20 μl of a suspension containing the crypts were placed directly on glass objective slides together with a glass coverslip. Images were acquired
with a 10x and a 40x objective and a digital camera attached to the microscope. A higher magnification of the field in the dotted squared is shown in the right panel. Crypts are in single or cluster elongated columnar and spherical cells.


Figure 3. Characterisation of CD45+ cells in mouse colon. Colon crypt cells from mouse were stained with anti-CD45, -CD4, CD8, -Ly6G and -F4/80 antibodies and the percentage of positive cells analysed by flow cytometry. Representative profiles are shown. Other FACS analysis and quantification image examples are published in Cancer Research (Del Reino et al., 2014). For details, refer to Figure 5B.

Recipes

  1. Phosphate-buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCI
    4.3 mM Na3PO4
    1.4 mM K2HPO4
  2. Disaggregation medium
    HBSS
    0.4 mg/ml dispase
    Penicillin/streptomycin: 100 U/ml penicillin and 0.1 mg/ml streptomycin

Acknowledgments

This work was supported by The Spanish Ministry of Economy and Competiveness (MINECO) (SAF2013-45331-R) and La Marató TV3 Foundation (82031).

References

  1. Clevers, H. (2013). The intestinal crypt, a prototype stem cell compartment. Cell 154(2): 274-284.
  2. Del Reino, P., Alsina-Beauchamp, D., Escos, A., Cerezo-Guisado, M. I., Risco, A., Aparicio, N., Zur, R., Fernandez-Estevez, M., Collantes, E., Montans, J. and Cuenda, A. (2014). Pro-oncogenic role of alternative p38 mitogen-activated protein kinases p38gamma and p38delta, linking inflammation and cancer in colitis-associated colon cancer. Cancer Res 74(21): 6150-6160.
  3. Mowat, A. M. and Agace, W. W. (2014). Regional specialization within the intestinal immune system. Nat Rev Immunol 14(10): 667-685.

简介

肠上皮层形成管状内陷到固有层的下面的结缔组织。 这些结构,称为隐窝,是肠的基本功能单位。 结肠隐窝和周围固有层存在不同的细胞类型,包括上皮细胞,干细胞,肠细胞,杯状细胞,以及先天和适应性免疫系统的细胞(Clevers,2013; Mowat和Agace,2014)。 在这里我们描述了用于分离小鼠肠道隐窝细胞的技术以及它们通过流式细胞术分析(FACS)的表征(Del Reino等人,2012)。

关键字:流式细胞术, 结肠, 肠, 腺窝细胞, 小鼠

材料和试剂

  1. Falcon 50ml锥形管(VWR International,目录号:21008-240)
  2. Falcon 15ml锥形管(VWR International,目录号:21008-216)
  3. 10cm培养皿(Corning,Falcon ,目录号:353046)
  4. 纱,筛目尺寸约100μm(Tegosa,目录号:10018)
  5. 细胞过滤器(70μm)(Corning,Falcon ,目录号:352350)
  6. 细胞过滤器(40μm)(Corning,Falcon ,目录号:352340)
  7. 25G针(Premier Healthcare& Hygiene,BD Microlance,目录号:300600)
  8. 5ml注射器(BD,目录号:309649)
  9. 5ml圆底流式细胞术管(BD Biosciences,目录号:352063)
  10. 8至10周龄小鼠()(男性或女性)(我们使用C57BL/6株)
  11. Hank's平衡盐溶液培养基(HBSS)(Life Technologies,Gibco ,目录号:2420-091)
  12. 青霉素 - 链霉素溶液100x(Biowest,目录号:L0022)
  13. 乙二胺四乙酸(EDTA)(AppliChem GmbH,Panreac,目录号:13102/131026)
  14. 胎牛血清(FBS)(Life Technologies,目录号:GCS0103-500)
  15. APC结合的抗CD45抗体(Beckman Coulter,目录号:732158)
  16. FITC标记的抗CD4抗体(Beckman Coulter,目录号:731999)
  17. PE缀合的抗Ly6G抗体(Beckman Coulter,目录号:732487)
  18. 生物素化的抗F480抗体(eBioscience,目录号:13-4801)
  19. PeyC7缀合的抗CD8抗体(Biolegend,目录号:100721)
  20. ECD缀合的链霉抗生物素蛋白(Beckman Coulter,目录号:IM3326)
  21. Dispase II(Life Technologies,目录号:17105-041)
    注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:17105-041"。
  22. 磷酸盐缓冲盐水(PBS)(见配方)
  23. 分解介质(参见配方)

设备

  1. 解剖设备(钳子和剪刀)
  2. 手术刀或剃刀刀片
  3. 37℃室(Thermo Fisher Scientific,Heraeus )
  4. 冷冻离心机(Thermo Fisher Scientific,Heraeus ,型号:Megafuge TM 2.012)
  5. Neubauer室细胞计数器
  6. 轨道振动平台(KühnerAG,Lab-Shaker)
  7. CO 2室
  8. Cytomics fc500流式细胞仪(Beckman Coulter)
  9. 显微镜(ZEISS,型号:Axiovert 40CFL)

程序

  1. 肠道隐窝细胞的分离
    1. 由受过训练的人员在CO 2室中牺牲小鼠。执行 从构造的腔室中的加压罐吸入CO 2 透明材料(如 Plexiglas)(标准尺寸的小鼠笼可能包含 不超过5只小鼠),随后颈椎脱臼。根据 机构动物护理和使用委员会,CO <2>必须在a 以每小时10-30%体积排量的低流速控制 分钟。
    2. 用酒精擦拭腹部后,做腹部 切口。使用镊子和剪刀仔细清除整个结肠 (图1)。从盲肠切到肛门(8-10厘米之间,取决于 ?小鼠大小),并将其放置在具有15ml冷PBS的10cm培养皿中 ?覆盖结肠。
    3. 保持结肠的远端部分 镊子和冲洗去除粪便与5-10毫升冷无菌PBS使用a 25 G针连接到5ml注射器。冲洗出粪便后,放置 清洁的结肠在一个新的培养皿中用10毫升冷PBS
    4. 在结肠纵向切口,用冷PBS再次冲洗,以清除残留的粪便
    5. 将开放的结肠放在冷的HBSS培养基在冰上,直到所有的冒号 ?如在步骤A1-4中那样提取实验。对于好的单元格 产量,我们在每个测试条件下使用三只小鼠
    6. 将所有结肠孵育在含有100U/ml青霉素和0.1mg/ml链霉素的5ml HBSS中(室温15分钟)。
    7. 剁碎结肠组织成小块约。 2mm×2mm ?培养皿,在手术刀的帮助下。把小块放在50 ml Falcon管中,并在5ml的8mM EDTA的HBSS(160μl0.5M)中孵育 EDTA在10ml HBSS中; 15分钟,37℃)
    8. 取出培养基,加入5 ml新鲜冷HBSS到猎鹰管底部的结肠碎片。
    9. 通过剧烈手摇分离隐窝上皮细胞以获得 ?富集隐窝的上清液。从组织碎片上清液 ?进入新的Falcon 50ml管中。向样品中加入新鲜冷HBSS 重复摇动步骤。重复此过程3-5次增加 地穴富集
    10. 为了确认隐窝分离,在显微镜下观察20μl上清液样品(图2)。
    11. 离心上清液(200×g/g,15分钟,4℃)
    12. 除去上清液。向沉淀中加入5ml的解聚 培养基,将其转移到15ml Falcon管中并孵育(30分钟,37℃ ?以2500rpm,轨道振荡) ?单细胞悬液。
    13. 解聚后,向培养基中加入250μlFBS(终浓度5%)以终止分散酶反应
    14. 依次通过100μm纱布,70和40μm细胞过滤器过滤细胞
    15. 通过离心(150×g,10分钟,4℃)收集细胞, 重悬于1.5ml染色缓冲液(PBS中的2%FBS)并计数活细胞 ?在细胞计数器中。来自三只小鼠的预期细胞产量为约。 2 ?x 10 7 cells。
    16. 染色1×10 6个肠道隐窝细胞 荧光标记的抗体对感兴趣的细胞标记物 标准FACS分析程序,并在流式细胞仪上分析。

  2. 流式细胞分析的实例
    1. 在200μl染色缓冲液(2%)中孵育1×10 6个肠道隐窝细胞, FBS在PBS中)与荧光标记的抗CD45,-CD4,-F4/80,-CD8和 ?-Ly6G抗体(20分钟,4℃,避光)。稀释 抗体。 4-CD45-APC,稀释1/100; CD4-FITC稀释1/100; F4/80-稀释1/200的生物素;稀释1/200的CD8-PeyC7和稀释的Ly6G-PE ?1/100。
    2. 用150μl染色缓冲液洗涤两次,离心(150×g,5分钟,4℃)并弃去上清液。
    3. 孵育细胞与稀释1:50(20分钟,4°C,避光)链霉亲和素
    4. 重复洗涤步骤B2。
    5. 将细胞重悬在200μl染色缓冲液中,并转移到具有220μl染色缓冲液的圆底流式细胞仪管中。
    6. 使用Kaluza程序分析染色,对CD45 +细胞中的每种抗体门控所有阳性细胞(图3)。

代表数据


图1.结肠分离的顺序图像按照文本中所述顺序获取图片。


图2.小鼠结肠隐窝的光学显微镜图像。将20μl含有隐窝的悬浮液与玻璃盖玻片一起直接放置在玻璃目标载玻片上。使用10x和40x物镜和连接到显微镜的数码相机获取图像。在右图中示出了以虚线平方表示的场的更高放大率。地穴是单个或群集的细长柱状和球形细胞

图3.小鼠结肠中CD45 +细胞的表征。小鼠的结肠crypt细胞用抗CD45,-CD4,CD8,-Ly6G和-F4/80染色抗体和通过流式细胞术分析的阳性细胞的百分比。显示了代表性的曲线。其他FACS分析和定量图像实例在Cancer Research(Del Reino等人,2014)中公开。有关详细信息,请参阅图5B。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl 2.7mM KCl
    4.3mM Na 3 PO 4 4/v/v 1.4mM K 2 HPO 4
  2. 分解介质
    HBSS
    0.4 mg/ml分泌酶
    青霉素/链霉素:100U/ml青霉素和0.1mg/ml链霉素

致谢

这项工作由西班牙经济和竞争力部(MINECO)(SAF2013-45331-R)和LaMaratóTV3基金会(82031)支持。

参考文献

  1. Clevers,H.(2013)。 肠道隐窝,原型干细胞区室 细胞 154(2):274-284。
  2. Del Reino,P.,Alsina-Beauchamp,D.,Escos,A.,Cerezo-Guisado,MI,Risco,A.,Aparicio,N.,Zur,R.,Fernandez-Estevez,M.,Collantes, ,Montans,J。和Cuenda,A。(2014)。 替代p38丝裂原活化蛋白激酶p38gamma和p38delta的致癌作用,连接炎症和癌症结肠炎相关的结肠癌。 Cancer Res 74(21):6150-6160。
  3. Mowat,A.M.和Agace,W.W。(2014)。 肠道免疫系统内的区域专业化。 Nat Rev Immunol 14(10):667-685。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alsina-Beauchamp, D., Reino, P. d. and Cuenda, A. (2015). Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells. Bio-protocol 5(21): e1635. DOI: 10.21769/BioProtoc.1635.
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