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Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages
制备巯基乙酸盐诱导的腹水巨噬细胞并测量IL-4刺激后的精氨酸酶活性   

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Abstract

Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by type 2 cytokines such as IL-4 and it is characterized by the regulation of the L-arginine metabolism. M2 macrophages convert arginine to ornithine and urea through the action of Arginase-1. Here we described a method for the isolation of peritoneal macrophages from thioglycollate-elicited mice and alternative activation by stimulation with IL-4. Intraperitoneal injection of thioglycollate elicits large numbers of macrophages into peritoneal cavity.

Keywords: Macrophages(巨噬细胞), Thioglycollate(巯基乙酸), Arginase(精), IL-4(IL-4)

Materials and Reagents

  1. Mice C57BL/6 male (age 8-12 weeks)
  2. Difco Fluid Thioglycollate medium (BD, catalog number: 225650 )
  3. 70% and 100% ethanol
  4. PBS (Lonza, catalog number: BE 17-515Q )
  5. RPMI 1640 (Lonza, catalog number: BE 12-115F )
  6. FBS (endotoxin<10 EU/ml) (Hyclone, catalog number: SV30160.03 )
  7. Penicillin-Streptomycin (Lonza, catalog number: BE 17-602E )
  8. Murine IL-4 (Peprotech, catalog number: 214-14 )
  9. Tris (Panreac Applichem, catalog number: 131940.1211 )
  10. NaCl (Merck KGaA, catalog number: 1.06404.1000 )
  11. EDTA (Sigma-Aldrich, catalog number: ED255 )
  12. Triton X-100 (Sigma-Aldrich, catalog number: 8787 )
  13. Protease inhibitor mixture (Sigma-Aldrich, catalog number: P8340 )
  14. Urea (Sigma-Aldrich, catalog number: 4883 )
  15. MnCl2 (Sigma-Aldrich, catalog number: 244589 )
  16. L-arginine monohydrochloride (Sigma-Aldrich, catalog number: A6969 )
  17. H2SO4 (Sigma-Aldrich, catalog number: 320501 )
  18. H3PO4 (Sigma-Aldrich, catalog number: 345245 )
  19. Isonitrosopropiophenone (Sigma-Aldrich, catalog number: I3502 )
  20. 12-well plates
  21. 1.5 ml Eppendorf tubes
  22. 50 ml sterile Falcon tubes
  23. Cell strainer (Becton Dickinson, catalog number: 352340 )
  24. 10 ml syringe
  25. 21G Needles
  26. 25G needles
  27. Lysis buffer (see Recipes)
  28. Heat-inactivated FBS (see Recipes)
  29. Stopping solution (see Recipes)

Equipment

  1. Bench-top refrigerated centrifuge
  2. Scissors
  3. Incubator (37 °C, 5% CO2 and 95 % humidity)
  4. Tissue culture hood (biosafety cabinet)
  5. Optical microscopy
  6. Neubauer cell counting chamber
  7. Espectrophotometer plate reader
  8. Thermoblock (Eppendorf Thermomixer Compact) or water bath

Procedure

  1. Injection of thyoglicollate into the peritoneum
    1. Preparation of 3% thioglycollate medium.
      1. Suspend 30 grams of thioglycollate medium in 1,000 ml of pyrogen-free water.
      2. Aliquot to 100 ml sterile bottles.
      3. Autoclave (15 psi/121 °C/15 min).
      4. After cooling, store in a dark place at room temperature for 2 months before using.
        Note: Thioglycollate solution needs to age for several weeks until it turns to brown in color. This process is important to increase the extravasation of peritoneal cells.
    2. Inject 2.5 ml of 3% thioglycollate medium i.p. per mouse using a 10 ml syringe with a 25 G needle. Wait for 4 days and harvest peritoneal cells (see step B).

  2. Isolation and culture of thioglycollate elicited peritoneal macrophages
    1. Sacrifice mouse with CO2 or isofluorane (the use of cervical dislocation is not indicated in this protocol in order to avoid potential internal bleeding).
    2. Clean abdomen with 70% ethanol.
    3. Remove skin to expose the peritoneal wall. Practice a small incision in the skin and pull firmly.
    4. Inject 10 ml of RPMI 1640 into the peritoneal cavity with a 25 G needle.
    5. Massage abdomen for approximately 30 sec.
    6. Recover peritoneal fluid as much as possible using a 10 ml syringe with a 21 G needle. (Usually approximately 8-10 ml fluid can be recovered from one mouse.)
    7. Remove needle from syringe and put fluid into a 50 ml conical centrifuge tube on ice.
    8. Centrifuge peritoneal cells 5 min at 300 x g (1,500 rpm, 4 °C). Discard the supernatant and collect the pellet.
    9. Resuspend cell pellet in 10 ml of RPMI 1640 medium and count cells. (Usually approximately 30-50 million cells can be recovered from one mouse.)
    10. Culture peritoneal cells (1 x 106/cm2) in a 12-well plate in 1 ml of RPMI containing 10% heat-inactivated FBS at 37 °C with 5% CO2 for 3 h.
    11. Remove non-adherent cells by extensive washing with RPMI containing 10% heat-inactivated FBS at 37 °C.
    12. Proceed to overnight cell starvation in the presence of 1 ml of RPMI 2% FBS per well.

  3. M2 activation of peritoneal macrophages
    1. After cell starvation, maintain the cells in RPMI 2% FBS and stimulate macrophages by adding IL-4 (20 ng/ml) to the 12-well plate and incubate at 37 °C for 24 h.

  4. Arginase activity measurement
    1. For cell dissociation, wash cells once with PBS and add an appropriate volume of lysis buffer at RT (see Recipes).
      Note: Usually 200 μl in each 12-well plate.
    2. Remove cells with a cell scraper, collect them into a 1.5 ml Eppendorf and pipette up and down 5 times with a 200 µl tip for complete suspension.
    3. Lyse the cells for 15 min at 4 °C.
    4. Then spin down cell lysates at full speed (10 min, 14,000 rpm, 16,800 x g) at 4 °C and isolate the supernatant. Store at 4 °C before use.
    5. Meanwhile, prepare a stock of 8 M urea in 50 mM Tris-HCl (pH 7.5).
    6. Dilute stock of urea in 50 mM Tris-HCl (pH 7.5) to yield a standard range from 25 to 1,500 μg/ml. (e.g. 25, 50 100, 250, 500, 1,000 and 1,500 μg/ml)
    7. Transfer 50 μl of cell lysates and standards to a 2 ml Eppendorf tube and add 50 μl of 10 mM MnCl2 diluted in 50 mM Tris-HCl (pH 7.5).
    8. Incubate tubes in a thermoblock or water bath for 10 min at 55 °C to trigger arginase-1 activity.
    9. Then add 50 μl of 0.5 M L-arginine to the tubes and incubate in a thermoblock or water bath at 37 °C for 60 min. This step induces arginine hydrolysis.
    10. Stop the reaction by adding 400 μl of stopping solution (H2SO4/H3PO4/H2O = 1/3/7, v/v/v).
    11. Next, add 50 μl of 9% isonitrosopropiophenone in 100% ethanol to each sample and standard, and incubate the tubes in a thermoblock at 100 °C for 60 min.
    12. Place the tubes in the dark at RT for 30 min.
    13. Transfer 100 μl/well of samples and standards in triplicate to a 96-well plate and read optical density at 540 nm with a 690 nm correction.
    14. Calculate sample concentrations from the standard curve and converted to Arginase Units using the following formula: [Urea Produced (μg/ml)/Total Protein (μg/ml)].

Recipes

  1. Lysis buffer
    20 mM Tris (pH 7.5)
    150 mM NaCl
    2 mM EDTA
    0.1% Triton X-100
    Protease inhibitor mixture 1 μg/ml
  2. Heat-inactivated FBS
    Inactivate FBS at 55 °C for 30 min
    Stored in aliquots at -20 °C
  3. Stopping solution (for 11 ml)
    H2SO4 1 ml
    H3PO4 3 ml
    H2O 7 ml

Acknowledgments

This study was supported by grant PI11.0036 from the FIS and MPY 1410/09 from ISCIII to SH, and by grant TPY-M-1068/13 to AL. AL was supported by MINECO-ISCIII, through the Miguel Servet Programme (CP12/03087). L. J-G. was supported by FIS (FI12/00340). This protocol is adapted from Jimenez-Garcia et al. (2015).
Note: The study was performed under an ISCIII approved protocol.

References

  1. Jiménez-García, L., Herránz, S., Luque, A. and Hortelano, S. (2015). Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages. Eur J Immunol 45(1): 273-286.

简介

巨噬细胞是在炎症过程中起重要作用的先天免疫的必需细胞群体。 已经描述了具有相反活性的两种主要不同表型:经典激活的巨噬细胞(M1)和交替激活的巨噬细胞(M2)。 小鼠巨噬细胞的备选激活可以由2型细胞因子例如IL-4诱导,并且其特征在于L-精氨酸代谢的调节。 M2巨噬细胞通过精氨酸酶-1的作用将精氨酸转化为鸟氨酸和尿素。 在这里我们描述了从巯基乙酸诱发小鼠分离腹膜巨噬细胞和IL-4刺激的替代激活的方法。 腹膜内注射巯基乙酸盐引发大量巨噬细胞进入腹膜腔。

关键字:巨噬细胞, 巯基乙酸, 精, IL-4

材料和试剂

  1. 小鼠C57BL/6雄性(年龄8-12周)
  2. Difco Fluid硫代乙醇酸盐培养基(BD,目录号:225650)
  3. 70%和100%乙醇
  4. PBS(Lonza,目录号:BE17-515Q)
  5. RPMI 1640(Lonza,目录号:BE 12-115F)
  6. FBS(内毒素<10EU/ml)(Hyclone,目录号:SV30160.03)
  7. 青霉素 - 链霉素(Lonza,目录号:BE17-602E)
  8. 鼠IL-4(Peprotech,目录号:214-14)
  9. Tris(Panreac Applichem,目录号:131940.1211)
  10. NaCl(Merck KGaA,目录号:1.06404.1000)
  11. EDTA(Sigma-Aldrich,目录号:ED255)
  12. Triton X-100(Sigma-Aldrich,目录号:8787)
  13. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  14. 脲(Sigma-Aldrich,目录号:4883)
  15. MnCl 2(Sigma-Aldrich,目录号:244589)
  16. L-精氨酸一盐酸盐(Sigma-Aldrich,目录号:A6969)
  17. H 2 SO 4(Sigma-Aldrich,目录号:320501)
  18. H sub 3 PO 4(Sigma-Aldrich,目录号:345245)
  19. 异丙基苯丙酮(Sigma-Aldrich,目录号:I3502)
  20. 12孔板
  21. 1.5 ml Eppendorf管
  22. 50ml无菌Falcon管
  23. 细胞过滤器(Becton Dickinson,目录号:352340)
  24. 10毫升注射器
  25. 21G针
  26. 25G针
  27. 裂解缓冲液(见配方)
  28. 热灭活FBS(参见配方)
  29. 停止解决方案(参见配方)

设备

  1. 台式冷冻离心机
  2. 剪刀
  3. 培养箱(37℃,5%CO 2和95%湿度)
  4. 组织培养罩(生物安全柜)
  5. 光学显微镜
  6. Neubauer细胞计数室
  7. 分光光度计读数器
  8. 热块(Eppendorf Thermomixer Compact)或水浴

程序

  1. 将甲糖醇注入腹膜
    1. 制备3%巯基乙酸盐培养基。
      1. 将30克巯基乙酸盐培养基悬浮在1000ml无热原水中
      2. 等分到100毫升无菌瓶。
      3. 高压灭菌(15 psi/121°C/15 min)。
      4. 冷却后,在室温下避光保存2个月后使用。
        注意:硫代乙醇酸盐溶液需要老化几个星期,直到它 颜色变为棕色。 这个过程是重要的增加 腹膜细胞外渗。
    2. 注入2.5ml的3% 巯基乙酸盐培养基i.p. 每只小鼠使用具有25G的10ml注射器 针。 等待4天,收获腹膜细胞(见步骤B)。

  2. 巯基乙酸钠的分离和培养引发腹膜巨噬细胞
    1. 牺牲小鼠与CO 2或异氟烷(使用颈脱位   不在本协议中指示,以避免潜在的内部 出血)。
    2. 用70%乙醇清洁腹部。
    3. 去除皮肤暴露腹膜壁。 在皮肤上做一个小切口,并牢固地拉紧
    4. 用25 G针将10ml RPMI 1640注入腹膜腔
    5. 按摩腹部约30秒。
    6. 使用10毫升注射器尽可能多的恢复腹膜液 用21 G针。 (通常约8-10ml液体即可 从一只老鼠恢复。)
    7. 从注射器中取出针头,将液体放入冰上的50ml锥形离心管中
    8. 在300×g(1,500rpm,4℃)下离心腹膜细胞5分钟。 弃去上清液并收集沉淀。
    9. 重悬细胞沉淀在10 ml RPMI 1640培养基和计数细胞。 (通常可以从一个回收约3,000-50,000,000个细胞 鼠标。)
    10. 在12孔培养物中培养腹膜细胞(1×10 6个/cm 2/cm 2) 平板在1ml含有10%热灭活的FBS的RPMI中 5%CO 2 3小时。
    11. 通过用含有10%热灭活的FBS的RPMI在37℃下彻底清洗去除非贴壁细胞
    12. 在每孔1ml RPMI 2%FBS存在下进行过夜细胞饥饿。

  3. M2激活腹膜巨噬细胞
    1. 细胞饥饿后,保持细胞在RPMI 2%FBS和刺激 通过向12孔板中加入IL-4(20ng/ml)并孵育来诱导巨噬细胞 在37℃下培养24小时。

  4. 精氨酸酶活性测量
    1. 对于细胞解离,用PBS洗涤细胞一次,并在室温下加入适当体积的裂解缓冲液(参见Recipes)。
      注意:通常在每个12孔板中200μl。
    2. 用细胞刮刀除去细胞,收集到一个1.5毫升 Eppendorf和移液器上下5次用200μl提示完成   悬挂。
    3. 在4℃下裂解细胞15分钟。
    4. 然后旋转   在4℃下以全速(10分钟,14,000rpm,16,800×g)洗涤细胞裂解物 并分离上清液。 使用前保存于4°C。
    5. 同时,在50mM Tris-HCl(pH7.5)中制备8M尿素储备液
    6. 稀释尿素在50mM Tris-HCl(pH7.5)中的储备液以产生标准物 范围为25至1,500μg/ml。 (例如25,50,100,250,500,1,000和1,500)   μg/ml)
    7. 转移50微升的细胞裂解液和标准品2毫升 Eppendorf管中,加入50μl在50mM Tris-HCl中稀释的10mM MnCl 2 (pH7.5)。
    8. 孵育管在热块或水浴中在55°C下10分钟,以触发精氨酸酶-1活性。
    9. 然后向管中加入50μl的0.5M L-精氨酸并在37℃下孵育 热块或在37℃水浴60分钟。 这个步骤诱导 精氨酸水解
    10. 通过加入400μl终止溶液(H 2 SO 4 SO 4/H 3 PO 4/H)终止反应 O = 1/3/7,v/v/v)。
    11. 接下来,加入50μl的9%异壬基苯丙酮在100%乙醇中 每个样品和标准品,并在100℃的热块中孵育试管   ℃60分钟。
    12. 将试管在黑暗中室温放置30分钟。
    13. 转移100微升/孔的样品和标准品一式三份到a 96孔板,在540nm用690nm读取光密度 更正。
    14. 根据标准计算样品浓度 曲线并使用下式转化为精氨酸酶单位:[尿素   产生的(μg/ml)/总蛋白(μg/ml)]。

食谱

  1. 裂解缓冲液
    20mM Tris(pH7.5) 150mM NaCl 2mM EDTA 0.1%Triton X-100 蛋白酶抑制剂混合物1μg/ml
  2. 热灭活的FBS
    在55℃下使FBS失活30分钟
    以等分试样储存在-20℃下
  3. 停止溶液(11 ml)
    H SO 1 ml
    H 3 4 3 ml
    H 2 O 7ml

致谢

这项研究由来自FIS的授权PI11.0036和来自ISCIII到SH的MPY1410/09以及通过授予TPY-M-1068/13到AL支持。 AL由MINECO-ISCIII通过Miguel Servet计划(CP12/03087)提供支持。 L.J-G。 由FIS(FI12/00340)支持。 该协议改编自Jimenez-Garcia等人(2015)。
注意:根据ISCIII批准的方案进行研究。

参考文献

  1. Jiménez-García,L.,Herránz,S.,Luque,A。和Hortelano,S。(2015)。 p38 MAPK在IL-4诱导的腹膜巨噬细胞的替代活化中的关键作用。 Eur J Immuno 45(1):273-286。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jiménez-García, L., Herránz, S., Luque, A. and Hortelano, S. (2015). Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages. Bio-protocol 5(17): e1585. DOI: 10.21769/BioProtoc.1585.
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