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Harvest and Culture of Mouse Peritoneal Macrophages
小鼠腹水中巨噬细胞的采集和培养   

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Abstract

Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited peritoneal cells. Peritoneal macrophages are non-adherent in situ and when they are cultured in dishes, they become adherent so that macrophages may be separated from other types of cells in peritoneal cavity.

Materials and Reagents

  1. C57BL/6 mouse
  2. 70% alcohol or isopropanol
  3. PBS (Life Technologies, catalog number: 10010023 )
  4. EDTA (Life Technologies, catalog number: 15575-020 )
  5. RPMI 1640 (Cellgro®, catalog number: 10-041-CV )
  6. 10% heat-inactivated FBS (endotoxin < 0.06 EU/ml) (Hyclone, catalog number: SH30071.03HI )
  7. Penicillin-Streptomycin-Glutamine (Life Technologies, catalog number: 10378016 )
  8. Nonessential amino acids (Life Technologies, catalog number: 11140050 )
  9. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
  10. Hepes (suitable for cell culture) (Sigma-Aldrich, catalog number: H0887 )
  11. 2-mercaptoethanol (EMD Millipore, catalog number: ES-007-E )
  12. Thioglycollate medium (BD Biosciences, catalog number: 211716 )
  13. Pyrogen-free water (Hyclone, catalog number: SH30529.03 )
  14. cRPMI medium (see Recipes)

Equipment

  1. 5 ml syringe
  2. 12-well plate
  3. 15 ml sterile Falcon tubes
  4. Needles (20 G and 25 G)
  5. Centrifuge
  6. 37 °C, 5% CO2 cell culture incubator
  7. 300 ml flask with an aluminum foil lid
  8. Laboratory oven

Procedure

  1. Harvest and culture of resident peritoneal cells
    1. Fill 5 ml of PBS with 5 mM EDTA in a 5 ml syringe with a 25 G needle.
    2. Anesthetize and sacrifice mouse with CO2.
    3. Place mouse with abdomen up on paper towel in hood.
    4. Swab abdomen with 70% alcohol or isopropanol.
    5. Make a small incision in the center of the skin overlying the peritoneal wall.
      Note: Small incision is made on the skin to peel the skin off. Injection and extraction can be at different sites on peritoneal membrane.
    6. Firmly pull skin to expose the peritoneal wall.
    7. Insert the needle to peritoneal membrane; avoid inserting needle into guts or bladder. Inject 5 ml of PBS EDTA into peritoneal cavity.
    8. Massage abdomen for approximately 10-15 sec.
    9. Withdraw the needle slowly. Change the 25 G needle on the syringe to a 20 G needle.
    10. Use one hand to push the fluid to one side of the peritoneum. Using the other hand, insert needle to the side of cavity with plenty of fluid and withdraw the fluid from peritoneum. Avoid fat, gut or mesentery, which may clog the needle. Try to draw as much fluid as possible. Usually, approximately 4 - 4.5 ml fluid can be recovered from one mouse.
    11. Remove needle from syringe and dispense contents into a centrifuge tube on ice.
    12. Centrifuge peritoneal cells (300 x g; 3 min) and collect cell pellet. Usually about 2-4 million resident peritoneal cells can be recovered from one C57BL/6 mouse using this method and about 50% are peritoneal macrophages.
    13. Resuspend cell pellet from one mouse in 1 ml of cRPMI medium, count cells.
    14. Culture peritoneal cells in a 12-well plate, 2 million/well in 1 ml cRPMI at 37 °C with 5% CO2 for 6-18 h. During this time, peritoneal macrophages adhere to the plastic surface. The floating non-macrophages can then be washed away by adding and aspirating 0.5 ml cRPMI medium twice.
    15. The adherent macrophages are ready to use.

  2. Harvest and culture thioglycollate-elicited peritoneal cells
    Thioglycollate elicited peritoneal macrophages can be harvested and cultured in the same way as described in steps 1-15 with additional steps as shown below.
    1. Preparation of 3% thioglycollate medium.
      1. Heat a 300 ml flask with an aluminum foil lid (180-200 °C) in a laboratory oven for at least 18 h to get rid of endotoxin.
      2. Suspend 6 grams of thioglycollate medium in 200 ml of pyrogen-free water.
      3. Autoclave (15 psi/121 °C/15 min).
      4. After cooling, aliquot to 15 ml sterile Falcon tubes. Store in a dark place at room temperature for 2 months before using. We have found that thioglycollate medium stored at room temperature for up to 2 years can still be used.
    2. Inject 1 ml of aged thioglycollate i.p. per mouse. Wait for 4-5 days, harvest peritoneal cells. About 10 million macrophages can be recovered from one mouse.

    Note: The study was performed under an IACUC approved protocol (LCID 11E).

Recipes

  1. cRPMI medium
    RPMI 1640 with 10% heat-inactivated FBS (endotoxin < 0.06 EU/ml)
    2 mM L-glutamine
    100 μM of nonessential amino acids
    100 U/ml penicillin
    0.1 mg/ml streptomycin
    10 μM of sodium pyruvate
    25 mM Hepes, pH 7.4
    50 μM 2-mercaptoethanol

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, NIAID

References

  1. Lu, M., Varley, A. W. and Munford, R. S. (2013). Persistently active microbial molecules prolong innate immune tolerance in vivo. PLoS Pathog 9(5): e1003339.

简介

腹膜巨噬细胞在许多研究中用作初级巨噬细胞,主要是因为它们容易获得。 注射巯基乙酸盐肉汤i.p. 诱导炎症反应并引发大量的巨噬细胞。 该方案可用于收获驻留的或巯基乙酸盐诱发的腹膜细胞。 腹膜巨噬细胞是原位非粘附的,并且当它们在培养皿中培养时,它们变得粘附,使得巨噬细胞可以与腹膜腔中的其它类型的细胞分离。

材料和试剂

  1. C57BL/6小鼠
  2. 70%酒精或异丙醇
  3. PBS(Life Technologies,目录号:10010023)
  4. EDTA(Life Technologies,目录号:15575-020)
  5. RPMI 1640(Cellgro ,目录号:10-041-CV)
  6. 10%热灭活的FBS(内毒素<0.06EU/ml)(Hyclone,目录号:SH30071.03HI)
  7. 青霉素 - 链霉素 - 谷氨酰胺(Life Technologies,目录号:10378016)
  8. 非必需氨基酸(Life Technologies,目录号:11140050)
  9. 丙酮酸钠(Sigma-Aldrich,目录号:S8636)
  10. Hepes(适用于细胞培养)(Sigma-Aldrich,目录号:H0887)
  11. 2-巯基乙醇(EMD Millipore,目录号:ES-007-E)
  12. 硫代乙醇酸盐培养基(BD Biosciences,目录号:211716)
  13. 无热原水(Hyclone,目录号:SH30529.03)
  14. cRPMI介质(参见配方)

设备

  1. 5毫升注射器
  2. 12孔板
  3. 15 ml无菌Falcon管
  4. 针(20 G和25 G)
  5. 离心机
  6. 37℃,5%CO 2细胞培养箱中培养
  7. 带有铝箔盖的300ml烧瓶
  8. 实验室炉

程序

  1. 驻留腹膜细胞的收获和培养
    1. 填充5毫升PBS与5毫米EDTA的5毫升注射器与25 G针。
    2. 麻醉和牺牲鼠标与CO 2
    3. 将鼠标放在腹部的纸巾上
    4. 用70%酒精或异丙醇擦拭腹部。
    5. 在覆盖腹膜壁的皮肤中心做一个小切口。
      注意:在皮肤上做小切口以剥离皮肤。注射和提取可以在腹膜的不同部位。
    6. 牢牢拉皮肤露出腹膜壁。
    7. 将针插入腹膜;避免将针插入内胆或膀胱。注射5ml PBS EDTA腹膜腔。
    8. 按摩腹部约10-15秒。
    9. 缓慢抽出针头。将注射器上的25 G针更换为20 G针
    10. 用一只手将液体推到腹膜的一侧。使用另一只手,用大量流体将针插入腔的侧面,并从腹膜中抽出流体。避免脂肪,肠道或肠系膜,这可能会堵塞针头。尽量画出尽可能多的流体。通常,从一只老鼠可以回收约4 - 4.5毫升的液体
    11. 从注射器中取出针头,并将内容物分装到冰上的离心管中
    12. 离心腹膜细胞(300×g; 3分钟)并收集细胞沉淀。 通常使用这种方法可从一只C57BL/6小鼠中回收约2-4百万个驻留的腹膜细胞,约50%是腹膜巨噬细胞。
    13. 从1只小鼠的1ml cRPMI培养基中重悬细胞沉淀,计数细胞
    14. 在12孔板中培养腹膜细胞,在37℃,5%CO 2下,在2ml /孔的1ml cRPMI中培养6-18小时。 在此期间,腹膜巨噬细胞粘附在塑料表面。 然后通过加入和抽吸0.5ml cRPMI培养基两次来冲洗漂浮的非巨噬细胞
    15. 粘附的巨噬细胞可以使用。

  2. 收获和培养巯基乙酸盐诱发的腹膜细胞
    可以按照如步骤1-15所述的相同方法,收获硫代乙醇酸盐诱导的腹膜巨噬细胞并培养,如下所示。
    1. 制备3%巯基乙酸盐培养基。
      1. 在实验室烘箱中用带有铝箔盖(180-200℃)的300ml烧瓶加热至少18小时以除去内毒素。
      2. 将6克巯基乙酸盐培养基悬浮在200ml无热原水中
      3. 高压灭菌(15 psi/121°C/15 min)。
      4. 冷却后,等分至15ml无菌Falcon管。 在使用前在室温下避光保存2个月。 我们已经发现,在室温下储存2年的巯基乙酸盐培养基仍然可以使用
    2. 注射1ml老化的巯基乙酸盐i.p. 每只小鼠。 等待4-5天,收获腹膜细胞。 从一只老鼠可以回收约10万个巨噬细胞

    注意:根据IACUC批准的方案(LCID 11E)进行研究。

食谱

  1. cRPMI培养基
    具有10%热灭活的FBS(内毒素<0.06EU/ml)的RPMI 1640 2mM L-谷氨酰胺 100μM非必需氨基酸
    100 U/ml青霉素
    0.1mg/ml链霉素 10μM丙酮酸钠 25mM Hepes,pH7.4 50μM2-巯基乙醇

致谢

这项研究由NIH,NIAID的Intramural研究计划支持

参考文献

  1. Lu,M.,Varley,A.W.and Munford,R.S.(2013)。 持续活性微生物分子在体内延长先天性免疫耐受性 。 PLoS Pathog 9(5):e1003339。
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Copyright: © 2013 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. Lu, M. and Varley, A. W. (2013). Harvest and Culture of Mouse Peritoneal Macrophages. Bio-protocol 3(22): e976. DOI: 10.21769/BioProtoc.976.
  2. Lu, M., Varley, A. W. and Munford, R. S. (2013). Persistently active microbial molecules prolong innate immune tolerance in vivo. PLoS Pathog 9(5): e1003339.
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wang liang
zhejiang hospital
Hi,Thank you for posting this protocol.
I gotta a question about Procedure B. After procedure B,what types of macrophage can we get ? M0,M1 or M2?
Thank you for answering my question,

Liang
7/13/2016 8:03:47 PM Reply