搜索

Adoptive Transfer of Isolated Bone Marrow Neutrophils
骨髓中性粒细胞的分离和移植   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Adoptive transfer experiments of specific cell populations are widely used methods to assess the role of the injected population on an ongoing process. In the last years, new and unprecedented roles in the regulation of immune responses have been reported for neutrophils. The following protocol is to be used to isolate neutrophils from bone marrow and to inject them in an appropriate host to test the role of neutrophils during infection, inflammation or other pathological conditions.

Material and Reagents

  1. Mice: Donor 8-12 week-old mice and sex-matched receptors. We always used C57BL/6 mice but it should work for any mice strain.
  2. Red blood lysing buffer Hybri-Max (Sigma-Aldrich, catalog number: R7757 )
  3. PBS (Life Technologies, Gibco®, catalog number: 10010-023 )
  4. EDTA (Life Technologies, Gibco®, catalog number: 15575-020 )
  5. Fetal Bovine Serum (FBS) (PAA Laboratories GmbH, catalog number: A15-201 )
  6. Anti-Ly-6G Microbead kit (Miltenyi Biotec, catalog number: 130-092-332 )
  7. 0.4% Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  8. PE-Cy7 labeled anti mouse Ly-6G (eBioscience, catalog number: 25-5931 )
  9. FITC labeled anti mouse CD11b (eBioscience, catalog number: 11-0112 )
  10. Isoflurane
  11. Flushing/washing buffer (see Recipes)
  12. MACS buffer (modified) (see Recipes)

Equipment

  1. LS column (Miltenyi Biotec, catalog number: 130-042-401 )
  2. Laminar Flow Cabinet
  3. MACS separators
  4. Centrifuge Eppendorf 5810R (Eppendorf, model: 5811000.010)
  5. Centrifuge rotor for plates A-4-62 (Eppendorf, model: 5810709.008 )
  6. Optic microscope
  7. Counting Neubauer chamber
  8. Flow cytometer BD FACS Canto II
  9. Automatic pipettes (full range volumes)
  10. Sterile forceps and scissors
  11. Tips (full range volumes)
  12. Microtubes (1.5 ml)
  13. Tubes (15 and 50 ml)
  14. Culture dishes (45 mm)
  15. Conventional Insulin 1 ml syringes with detachable needle (25G) (BD Biosciences, catalog number: 329651 )
  16. Low-dead volume insulin 1 ml syringes (29G) (BD Biosciences, catalog number: 329410 )
  17. Cell strainer 40 μm (BD Biosciences, catalog number: 352340 )
  18. Bell jar
  19. 0.2 μm filter

Procedure

Work fast and always keep buffers and cell suspensions on ice. These will avoid neutrophil apoptosis and/or activation.

  1. Bone marrow cell isolation
    1. Euthanize donor WT mice by an approved method.
    2. Clip and remove the skin from the arms and legs.
    3. Remove as much as possible tissue from arms and legs using scissors and dissect the bones away from body. To this end, cut the arms at the levels of the shoulders and legs at the levels of the hips.
    4. Separate the feet and hands by delicately twisting the joints with two forceps. Work carefully to avoid breaking the bones. Do not separate knees and elbows at this step.
    5. Put the arms and legs on ice in 1.5 ml microtubes filled with washing buffer until all bones have been dissected. The washing buffer should completely cover the bones.
    6. Transfer the bones to a 45 mm culture dish. If there are too much remaining tissues in the bones, remove it using scissors or scalpels as cells associated to the remaining tissue may contaminate marrow preparation.
    7. Separate the bones from the arms and legs gently twisting the elbows and knees with two sterile forceps.
    8. Cut off each end of the bone using sterile scissors and forceps.
    9. Harvest the marrow from the bone by inserting a 25 g needle in one end of the bone and flushing 1 ml of washing buffer with the syringe into a 15 ml tube.
    10. Repeat with the other end of the bone. Repeat for all the bones to obtain a cell suspension (typically 15 ml tube can collect bone marrow from 2 mice). While flushing the marrow, the 15 ml tube can remain at room temperature, but work fast and once the tube is filled, transfer it to ice.
    11. Centrifuge cell suspension at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    12. Lyse red blood cell using 2.5 ml red blood lysing buffer per tube that contains the bone marrow cell pellet from 2 mice (approximately, 150-200 x 106 cells). Scale-up as required. Gentle pipet up and down to disaggregate any clump. Incubate 5 min at room temperature.
    13. Inactivate lysing buffer with 7.5 ml washing buffer and filter cell suspension into a 50 ml using a 40 μm cell strainer.
    14. Centrifuge cell suspension at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    15. Resuspend pellet in 5 ml of MACS buffer and count viable cell number using 0.4% Trypan blue solution, a Neubauer chamber and an optic microscope. Record total cell number. Set aside 2 x 105 cells for evaluating purification efficiency as described below.
    16. Centrifuge cell suspension at 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.

  2. Neutrophil purification from bone marrow using Miltenyi kit
    1. Resuspend the cell pellet in 200 μl of MACS buffer per 108 cells. Use total cell number determined in step A13 to scale-up the amounts of buffer, antibodies and beads.
    2. Add 50 μl of Anti-Ly-6G-Biotin per 108 total cells.
    3. Mix well and incubate for 10 min in the refrigerator (2-8 °C).
    4. Add 150 μl of MACS buffer per 108 total cells.
    5. Add 100 μl of Anti-Biotin MicroBeads per 108 total cells.
    6. Mix well and incubate for 15 min in the refrigerator (2-8 °C).
    7. Wash cells by adding 10 ml MACS buffer and centrifuge cell suspension at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    8. Resuspend up to 108 cells in 500 μl of buffer and proceed to magnetic separation.
    9. Place an LS column in a suitable MACS Separator and wash the column by rinsing with 3 ml of MACS buffer.
    10. Apply cell suspension from step B8 onto the column. Collect flow-through containing unlabeled cells.
    11. Wash the column three times with 3 ml of MACS buffer.
    12. Collect unlabeled cells that pass through and combine with the flow through from step B11. Centrifuge unlabeled cell suspension and resuspend in 5 ml washing buffer. Count viable cell number using 0.4% Trypan blue solution, a Neubauer chamber and an optic microscope. Set aside 2 x 105 cells for evaluating purification efficiency as described below.
    13. Remove the column from the separator and place it on a suitable collection tube (typically 15 ml tube).
    14. Pipette 5 ml of MACS buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
    15. Centrifuge cell suspension at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    16. Resuspend the cell pellet in 5 ml of washing buffer and keep on ice. Count viable cell number using 0.4% Trypan blue solution, a Neubauer chamber and an optic microscope. Record total cell number. Set aside 2 x 105 cells for evaluating purification efficiency as described below.

  3. Control of purification efficiency by flow cytometry
    1. Stain 2 x 105 total bone marrow cells (step A15), unlabeled bone marrow cells (step B12) and Ly-6G+ isolated bone marrow cell (step B16) with 20 μl of a suspension contained pre-titrated amounts of anti-mouse Ly-6G and anti-mouse CD11b.
    2. Incubate 10 min at 4 °C and wash in 150 μl of washing buffer.
    3. Centrifuge cell suspension at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    4. Resuspend in 200 μl of washing buffer and analyze in a flow cytometer.
    5. Purity of neutrophils (Ly-6G high CD11b high) in the Ly-6G+ population should be higher than 95%.
      Percentages of neutrophils (Ly-6G high CD11b high) in total bone marrow cells as well as in unlabeled cells together with cell counts will allow calculation of the purification yield. Typically our yields are around 50%.

  4. Neutrophil injection in the retro-orbital sinus
    1. Centrifuge cell suspension from step B16 at 4 °C and 2,000 rpm for 5 min. Discard supernatant and keep the cell pellet.
    2. Wash cell pellet twice in 5 ml PBS.
    3. Resuspend cell pellet in PBS to a density of 40 - 50 x 106 cells per ml.
    4. Anesthetize the mouse using 30% V/V isoflurane in propylene glycol mixture in a bell jar of a size appropriate for the size of the animal. Work inside a chemical fume hood.
    5. After removal from the anesthetic, place the mouse in lateral recumbancy with the eye to be injected facing up.
    6. Retract the skin above the eye toward the body causing the eye to protrude.
    7. Insert the needle bevel up into the medial canthus of the eye at a 45 °C angle to the nose into the vessels behind the eye ball. Insert the needle around 2-3 mm, avoiding reaching the bone behind the eye.
    8. Gently inject up to 100 μl (maximum volume allowed) of the neutrophil cell suspension into the retro-orbital vessels using low dead volume syringes (29G).
    9. Slowly withdraw the needle and apply light pressure to the eye to control bleeding.
    10. If properly performed, only small amount of blood or liquid should come out from the eye at the site of the injection. Liquid or blood should never come out from the nose. No inflammation of the eye or peri-orbital area should be observed.
    11. Only one injection of a maximum of 100 μl volume per day is allowed. If repeated adoptive transfer is required you should alternate the eyes for injection allowing at least one day of recovery and with a maximum of total 2 injections per eye (4 injections per mice).


      Figure 1. Retro-orbital sinus injection. The scheme shows the proper site of cell injection including the correct needle bevel placement.

Recipes

Prepare the following buffer in advance and keep at 4 °C throughout the procedure:

  1. Flushing/washing buffer
    2% FBS PBS
  2. MACS buffer (modified)
    2 mM EDTA
    2% FBS
    PBS (pH 8.2) filtered through a 0.2 μm filter

Acknowledgments

This work received financial support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), from the Agencia Nacional de Promoción Científica y Técnica and form the Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba. EAR is member of the scientific career of the Consejo Nacional de Investigaciones Científicas y Técnicas. JTB thank Consejo Nacional de Investigaciones Científicas y Técnicas for the fellowship awarded.

References

  1. Tosello Boari, J., Amezcua Vesely, M. C., Bermejo, D. A., Ramello, M. C., Montes, C. L., Cejas, H., Gruppi, A. and Acosta Rodriguez, E. V. (2012). IL-17RA signaling reduces inflammation and mortality during Trypanosoma cruzi infection by recruiting suppressive IL-10-producing neutrophils. PLoS Pathog 8(4): e1002658.

简介

特定细胞群体的过继转移实验是广泛使用的方法来评估注射群体对正在进行的过程的作用。 在过去几年中,已经报道了中性粒细胞在免疫应答的调节中的新的和前所未有的作用。 以下方案用于从骨髓中分离嗜中性粒细胞,并将其注射在合适的宿主中,以在感染,炎症或其它病理状况期间测试嗜中性粒细胞的作用。

材料和试剂

  1. 小鼠:供体8-12周龄小鼠和性别匹配的受体。 我们总是使用C57BL/6小鼠,但它应该适用于任何小鼠品系
  2. 红血球溶解缓冲液Hybri-Max(Sigma-Aldrich,目录号:R7757)
  3. PBS(Life Technologies,Gibco ,目录号:10010-023)
  4. EDTA(Life Technologies,Gibco ,目录号:15575-020)
  5. 胎牛血清(FBS)(PAA Laboratories GmbH,目录号:A15-201)
  6. 抗Ly-6G微珠试剂盒(Miltenyi Biotec,目录号:130-092-332)
  7. 0.4%台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  8. PE-Cy7标记的抗小鼠Ly-6G(eBioscience,目录号:25-5931)
  9. FITC标记的抗小鼠CD11b(eBioscience,目录号:11-0112)
  10. 异氟烷
  11. 冲洗/清洗缓冲液(见配方)
  12. MACS缓冲区(修改)(参见配方)

设备

  1. LS柱(Miltenyi Biotec,目录号:130-042-401)
  2. 层流柜
  3. MACS分隔符
  4. 离心机Eppendorf 5810R(Eppendorf,型号:5811000.010)
  5. 离心转子板A-4-62(Eppendorf,型号:5810709.008)
  6. 光学显微镜
  7. 计数Neubauer房间
  8. 流式细胞仪BD FACS Canto II
  9. 自动移液器(全量程)
  10. 无菌镊子和剪刀
  11. 提示(全范围卷)
  12. 微管(1.5ml)
  13. 管(15和50 ml)
  14. 培养皿(45毫米)
  15. 常规胰岛素具有可拆卸针(25G)的1ml注射器(BD Biosciences,目录号:329651)
  16. 低死体积胰岛素1ml注射器(29G)(BD Biosciences,目录号:329410)
  17. 细胞过滤器40μm(BD Biosciences,目录号:352340)
  18. 铃铛
  19. 0.2μm过滤器

程序

工作速度快,并始终保持缓冲液和细胞悬浮在冰上。 这些将避免中性粒细胞凋亡和/或活化。

  1. 骨髓细胞分离
    1. 安乐死供体WT小鼠通过批准的方法
    2. 夹住并清除手臂和腿部的皮肤。
    3. 使用剪刀从手臂和腿尽可能多的组织,并解剖骨头远离身体。为此,在臀部的肩膀和腿的水平处剪断手臂。
    4. 通过用两个镊子精细地扭转关节来分开脚和手。小心工作,以避免打破骨头。在此步骤中不要分离膝盖和肘部。
    5. 将手臂和腿放在冰上的1.5毫升微量管充满洗涤缓冲液,直到所有的骨已经解剖。洗涤缓冲液应完全覆盖骨骼。
    6. 将骨转移到45 mm培养皿。如果骨骼中剩余的组织太多,则使用剪刀或解剖刀将其去除,因为与剩余组织相关的细胞可能污染骨髓制备。
    7. 用两根无菌镊子轻轻扭曲肘部和膝盖,将胳膊和腿部的骨骼分开。
    8. 用无菌剪刀和镊子切除骨的每一端
    9. 通过在骨的一端插入25g针并用注射器将1ml洗涤缓冲液冲洗到15ml管中,从骨中收获骨髓。
    10. 重复与骨的另一端。对所有骨重复以获得细胞悬浮液(通常15ml管可从2只小鼠收集骨髓)。冲洗骨髓时,15毫升管可以保持在室温,但工作快,一旦管填充,将其转移到冰。
    11. 在4℃和2,000rpm离心细胞悬浮液5分钟。弃去上清液并保留细胞沉淀。
    12. 溶解红细胞,使用2.5ml红细胞溶解缓冲液/管,其含有来自2只小鼠(约150-200×10 6个细胞)的骨髓细胞沉淀。按需扩大。温和的移液器上下分解任何团块。在室温下孵育5分钟。
    13. 使用7.5ml洗涤缓冲液使过滤细胞悬浮液失活到50ml,使用40μm细胞过滤器。
    14. 在4℃和2,000rpm离心细胞悬浮液5分钟。 弃去上清液并保留细胞沉淀。
    15. 重悬沉淀在5ml MACS缓冲液中,并使用0.4%台盼蓝溶液,Neubauer室和光学显微镜计数活细胞数。 记录总细胞数。 放置2×10 5个细胞用于评估如下所述的纯化效率
    16. 离心细胞悬液在2,000 rpm 5分钟。 弃去上清液并保留细胞沉淀。

  2. 使用Miltenyi试剂盒从骨髓中纯化中性粒细胞
    1. 重悬细胞沉淀在200μl的MACS缓冲液每10 8个细胞。 使用步骤A13中确定的总细胞数量来放大缓冲液,抗体和珠子的量
    2. 每10个 8个总细胞加入50μl的抗Ly-6G-生物素
    3. 充分混合并在冰箱(2-8℃)中孵育10分钟
    4. 每10个 8个总细胞加入150μlMACS缓冲液。
    5. 每10个<8> 总细胞加入100μl抗生物素微珠
    6. 充分混合并在冰箱(2-8℃)中孵育15分钟
    7. 通过加入10ml MACS缓冲液洗涤细胞,并在4℃和2,000rpm离心细胞悬液5分钟。弃去上清液并保留细胞沉淀。
    8. 在500μl缓冲液中重悬多至10个细胞,并进行磁性分离
    9. 将LS柱放在合适的MACS分离器中,并用3ml MACS缓冲液冲洗柱子
    10. 将来自步骤B8的细胞悬浮液应用到柱上。收集含有未标记细胞的流过物
    11. 用3ml MACS缓冲液洗涤柱子三次。
    12. 收集未标记的细胞,其穿过并与来自步骤B11的流过组合。离心未标记的细胞悬液,并重悬于5ml洗涤缓冲液中。使用0.4%台盼蓝溶液,Neubauer室和光学显微镜计数活细胞数。放置2×10 5个细胞用于评估如下所述的纯化效率
    13. 从分离器中取出色谱柱,将其放在合适的收集管(通常为15 ml管)上
    14. 吸取5毫升MACS缓冲液到柱上。 通过将柱塞牢牢地推入柱中,立即冲出带有磁性标记的细胞
    15. 在4℃和2,000rpm离心细胞悬浮液5分钟。 弃去上清液并保留细胞沉淀。
    16. 重悬细胞沉淀在5ml的洗涤缓冲液,并保持在冰上。 使用0.4%台盼蓝溶液,Neubauer室和光学显微镜计数活细胞数。 记录总细胞数。 放置2×10 5个细胞用于评估如下所述的纯化效率

  3. 通过流式细胞术控制纯化效率
    1. 用20μl悬浮液包含预处理的细胞,染色2×10 5总骨髓细胞(步骤A15),未标记的骨髓细胞(步骤B12)和Ly-6G +分离的骨髓细胞(步骤B16) 滴定量的抗小鼠Ly-6G和抗小鼠CD11b
    2. 在4℃孵育10分钟,并在150μl洗涤缓冲液中洗涤
    3. 在4℃和2,000rpm离心细胞悬浮液5分钟。 弃去上清液并保留细胞沉淀。
    4. 重悬于200μl洗涤缓冲液中,并在流式细胞仪中分析
    5. Ly-6G +群体中嗜中性粒细胞(Ly-6G高CD11b高)的纯度应高于95%。
      在总骨髓细胞以及未标记细胞中的嗜中性粒细胞(Ly-6G高CD11b高)的百分比以及细胞计数将允许计算纯化产量。 通常我们的收益率为50%左右。

  4. 中性粒细胞注射在眼眶后窦
    1. 在4℃和2,000 rpm离心来自步骤B16的细胞悬液5分钟。 弃去上清液并保留细胞沉淀。
    2. 在5ml PBS中洗涤细胞沉淀两次
    3. 重悬细胞沉淀在PBS中,密度为每毫升40 - 50×10 6个细胞。
    4. 麻醉小鼠使用30%V/V异氟烷的丙二醇混合物在钟罩中大小适合于动物的大小。 在化学通风橱内工作。
    5. 从麻醉剂中取出后,将小鼠侧卧,用眼睛朝上注射。
    6. 将眼睛上方的皮肤朝向身体,使眼睛突出。
    7. 将针斜面向上进入眼睛的内侧眼角,以45°角与鼻子进入眼球后面的血管。将针头插入2-3毫米,避免接触眼睛后面的骨头。
    8. 使用低死体积注射器(29G)轻轻地注射高达100μl(允许的最大体积)嗜中性粒细胞悬浮液进入眼窝后血管。
    9. 慢慢撤回针头,并对眼睛施加轻微压力以控制出血。
    10. 如果正确执行,在注射部位只有少量的血液或液体从眼睛流出。液体或血液不应该从鼻子里出来。不应观察到眼睛或眶周区域的炎症。
    11. 只允许每天最多100μl体积的一次注射。如果需要重复的过继转移,您应该交替眼睛注射,允许至少一天的恢复和每眼最多总共2次注射(每只小鼠4次注射)。


      图1.眼眶后静脉注射。该方案显示了正确的细胞注射部位,包括正确的针斜角位置。

食谱

提前准备以下缓冲液,并在整个程序中保持在4°C:

  1. 冲洗/洗涤缓冲液
    2%FBS PBS
  2. MACS缓冲区(已修改)
    2mM EDTA 2%FBS
    PBS(pH8.2)通过0.2μm过滤器过滤

致谢

这项工作得到了儿童基金会/开发计划署/世界银行/世卫组织热带病研究和培训特别规划(TDR)的资金支持,来自科学和技术促进委员会,并成立了国家科学和技术大学 。 EAR是Consejo Nacional de InvestigacionesCientíficasyTécnicas的科学生涯的成员。 JTB感谢Consejo Nacional de InvestigacionesCientíficasyTécnicas获得奖学金。

参考文献

  1. Tosello Boari,J.,Amezcua Vesely,M.C.,Bermejo,D.A.,Ramello,M.C.,Montes,C.L.,Cejas,H.,Gruppi,A.and Acosta Rodriguez,E.V。(2012)。 IL-17RA信号通过募集抑制性IL-10产生嗜中性粒细胞减少锥虫感染期间的炎症和死亡率 。 PLoS Pathog 8(4):e1002658。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Boari, J. T. and Rodríguez, E. A. (2012). Adoptive Transfer of Isolated Bone Marrow Neutrophils. Bio-protocol 2(23): e298. DOI: 10.21769/BioProtoc.298.
  2. Tosello Boari, J., Amezcua Vesely, M. C., Bermejo, D. A., Ramello, M. C., Montes, C. L., Cejas, H., Gruppi, A. and Acosta Rodriguez, E. V. (2012). IL-17RA signaling reduces inflammation and mortality during Trypanosoma cruzi infection by recruiting suppressive IL-10-producing neutrophils. PLoS Pathog 8(4): e1002658.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。