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本实验方案简略版
Apr 2017

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Eicosanoid Isolation from Mouse Intestinal Tissue for ELISA
从小鼠肠道组织中分离类二十烷酸用于ELISA   

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Abstract

Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal tissue for analysis by PGE2-ELISA and normalize to tissue protein content.

Keywords: Intestine (肠道), Eicosanoid (类二十烷酸), ELISA (ELISA), Prostaglandin (前列腺素), Lipid extraction (脂类提取)

Background

Inflammasome induced eicosanoid release is a relatively recent observation. It is not clear which cell/tissue types other than peritoneal macrophages and intestinal epithelial cells (IEC) (Rauch et al., 2017) can release prostaglandins upon inflammasome activation yet. This protocol can be adapted for other types of tissue as well as measurement of eicosanoid release induced by other stimuli than inflammasome activation in intestinal tissue.

Note that this protocol is specifically for use of eicosanoid analysis by ELISA, other protocols have been described for eicosanoid analysis by lipid mass spectrometry.

Materials and Reagents

  1. Culture tubes (Corning, Falcon, catalog number: 352059)
  2. 15 ml tubes (Corning, Falcon, catalog number: 352097)
  3. Flat bottom 96-well plates, transparent (Corning, Costar, catalog number: 3370)
  4. pH test strips (GE Healthcare, Whatman, catalog number: 2613-991)
  5. SPE Cartridges (C-18) (Cayman Chemical, catalog number: 400020)
  6. Paper towel
  7. Liquid nitrogen
  8. UltraPureTM water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977)
  9. Indomethacin (Sigma-Aldrich, catalog number: I8280-5G)
  10. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010)
  11. 200 proof Kopec Ethanol (Decon Labs, catalog number: V1001)
  12. Methanol (Fisher Scientific, catalog number: A452SK)
  13. Ethyl acetate (Sigma-Aldrich, catalog number: 270989)
  14. Prostaglandin E2 ELISA Kit (Monoclonal) (Cayman Chemical, catalog number: 514010)
  15. BCA assay kit (Thermo Fisher Scientific, PierceTM, catalog number: 23227)
  16. K2HPO4 (Fisher Scientific, catalog number: P288)
  17. KH2PO4 (Fisher Scientific, catalog number: P285)
  18. DMSO (Sigma-Aldrich, catalog number: D8418)
  19. EDTA (Life Technologies, catalog number: 15575020)
  20. Sodium acetate (Sigma-Aldrich, catalog number: S2889)
  21. Acetic acid (Sigma-Aldrich, catalog number: A6283)
  22. Phosphate buffer (see Recipes)
  23. 1 M acetate buffer (see Recipes)

Equipment

  1. Forceps
  2. Scissors
  3. POLYTRON® PT 2500 E Stand Dispersion Unit (Ecoline) with 12 mm aggregate (Kinematica) or similar
  4. Fridge
  5. Centrifuge for 15 ml reaction tubes capable of cooling (Eppendorf 5810R or similar)
  6. Vortex
  7. 96-well plate reader capable of measuring absorbance between 405 and 420 nm and 562 nm (e.g., Molecular Devices, model: Spectramax® M2)
  8. Reacti-VapTM Evaporator (Thermo Fisher Scientific, catalog numbers: TS-18825 or TS-18826) or similar
  9. Nitrogen gas with pressure gauge hooked up to evaporator
  10. SPE Vacuum Manifold (Sigma-Aldrich, catalog number: 57250-U or similar)
  11. Vacuum trap (e.g., Fisher Scientific, FisherbrandTM Reusable Heavy-Wall Filter Flasks, catalog number: FB3001000) connected to central vacuum system or pump (e.g., Merck, Chemical Duty Vacuum Pressure Pump, catalog number: WP6122050) via tubing and rubber stopper

Procedure

Workflow:

Day 1:
Animal treatment and tissue harvest (1-4 h depending on number of animals)
Day 1 or 2:
Tissue homogenization and centrifugation (30-60 min)

Evaporation 1 (60-90 min)

Resuspend, pH, C-18 columns (30-60 min)

Evaporation 2 (60-90 min)

Resuspend, load ELISA (1-2 h)
Day 2 or 3:
Finish ELISA (2 h)

Perform BCA assay (1 h)


  1. Harvest intestinal tissue and immediately snap freeze in liquid nitrogen. To harvest intestine, cut open the peritoneum of sacrificed mouse, find stomach below lower end of the ribcage, separate small intestine from stomach and gently pull with forceps while separating from mesentery using scissors. Separate from anus and lay out to get an overview of the tissue. Make sure to harvest comparable areas of tissue from individual animals.
    Note: 2 cm of tissue is easily sufficient. Mice do not need starving previous to tissue isolation. It is sufficient to remove large amounts of intestinal content by moving forceps along the chosen piece of intestine on a paper towel and thus squeeze most of the content out.
  2. Homogenize tissue in 1 ml phosphate buffer/100 mg. Make sure tissue does not thaw before the homogenization step but drop frozen tissue directly into your homogenization-tube filled with buffer.
    Note: Only turn homogenizer on once the tip is immersed in fluid with tissue. Move sample slightly up and down while homogenizing without removing tip from fluid completely. Homogenize at appropriate speed and for appropriate time to completely homogenize tissue (e.g., speed 26-30 for 10 s on POLYTRON® PT 2500 E).
  3. Remove 30 µl aliquot of homogenate for BCA assay to determine protein concentration later, freeze (dilute 1:10 in PBS for assay: add 270 µl PBS to the 30 µl homogenate and mix).
  4. Pre-cool the centrifuge to 4 °C. To precipitate proteins, add 100% ethanol (four times the sample volume, e.g., with 1 ml homogenization buffer use 4 ml ethanol) to each tube. Vortex to mix thoroughly and put at 4 °C immediately. Incubate samples at 4 °C for 5 min, then centrifuge at 4 °C, 3,000 x g for 10 min to remove precipitated proteins. Transfer the supernatant to a clean test tube.
    Note: If you homogenized a large amount of tissue in a lot of buffer, only use a 1.5 ml aliquot for these next steps, otherwise the evaporation will take a very long time. Remember to include this in your back-calculations at the end. Keep the rest of the supernatant at -20 °C until you are finished with the isolation, can be used as a backup in case something goes wrong.
  5. Evaporate the ethanol under nitrogen to complete dryness (takes 60-90 min for 1.5 ml) 
    1. Use ~2 PSI pressure per 16 samples (increase or decrease pressure if more or less samples are dried), increase accordingly. Keep moving needles down onto samples to a distance so bubbling can be heard but no splashing is observed. See also Figure 1.


      Figure 1. Needle positioning during nitrogen drying. Surface rippling of the liquid should be observed, but no drops should be splashing to the sides of the tubes.

    2. Yellowish remnants in the tube are normal.
  6. Resuspend sample in UltraPureTM water and acidify to ~pH 4.0 by the addition of 1 M acetate buffer.
    Note: For your first sample, carefully add more acetate buffer as you go and note how much you need, then add the same amount to samples of the same type. For sample evaporated from 1.5 ml, resuspended in 300 µl, amount of acetate buffer needed is approximately 45 µl. Sample can also be resuspended in more water if desired, this will however increase column time in Step 9. For the first test of a certain sample type it is recommended to have more sample for pH testing. To check the pH of your sample using very small amounts, cut a pH test strip lengthwise to about 1/4 of the original width and use 5 µl distributed in drops onto the test fields. Record the amount of sample removed to include in back-calculations. If the samples contain precipitate, centrifuge to remove the precipitate. Particulate matter in the sample may clog the SPE Cartridge (C-18). 
  7. Put as many C-18 cartridges as samples onto vacuum manifold, close unused outlets. 
  8. Set vacuum on vacuum manifold to ~4. Prepare SPE (C-18) columns by rinsing with 5 ml methanol followed by 5 ml deionized water. (Drop speed should be ~2/sec). Do not allow the SPE Cartridge (C-18) to run dry until Step 10. The little valves below the cartridge can be used to adjust drop speed/stop cartridge if sample is in danger of running dry.
  9. Apply the sample to the SPE Cartridge (C-18) and allow the sample to completely enter the packing material. 
  10. Wash the column with 5 ml deionized water. Discard the wash. Let column run dry (increase vacuum to get completely dry).
  11. Elute the PGE2 from the column with 5 ml ethyl acetate containing 1% methanol. Make sure valves are from a material not sensitive to this solvent (or, if another type of vacuum manifold using stopcocks is used, remove them and let ethyl acetate enter column by gravity, elute with low vacuum.).
  12. Evaporate the ethyl acetate to dryness under a stream of nitrogen as described in Step 5. It is very important that all of the organic solvent be removed as even small quantities will adversely affect the ELISA (takes again about 60-90 min).
  13. To resuspend the sample, add 500 μl ELISA Buffer from the ELISA kit, no matter the initial volume. This amount ensures enough buffer in the kit is left for sample dilutions and preparation of other reagents. If more starting material was used, consider this in the sample dilution in Step 14. Vortex. It is common for insoluble precipitate to remain in the sample after addition of ELISA Buffer; this will not affect the assay. This sample is now ready for use in the ELISA. 
  14. Depending on expected PGE2 concentrations sample needs to be diluted in ELISA buffer. After systemic inflammasome activation, dilution of 1:100 is recommended.
  15. Perform PGE2 ELISA as described in Chayman chemicals protocol (https://www.caymanchem.com/pdfs/514010.pdf).
  16. Perform BCA assay on aliquots of tissue homogenate taken after homogenization of tissue according to kit protocol. Dilute tissue homogenate 1:10 in PBS for assay.
    Note: Make sure to also dilute standards in phosphate homogenization buffer/PBS 1/10.

Data analysis

Back-calculate sample PGE2 concentration according to the description in the ELISA protocol. Do not forget to account for taking only an aliquot of the spin supernatant.
Calculate the amount of protein in sample according to BCA assay.
Now you can calculate the ratio of PGE2/mg total protein in your sample.

Notes

  1. Load the samples onto the ELISA on the day of extraction, eicosanoids are very unstable, even at -80 °C.
  2. Depending on your tissue and eicosanoid production induction, you might have to test for different amounts of tissue used for isolation or different dilutions of sample used in the ELISA.

Recipes

  1. Phosphate buffer
    1. Stock: 133 g K2HPO4 + 32.15 g KH2PO4 in 1 L UltraPureTM water. The pH should be 7.4. 
    2. Dilute 1:10, add 1 mM EDTA + 10 µM Indomethacin right before use. Indomethacin needs to be dissolved fresh at 10 µM in DMSO
  2. 1 M acetate buffer
    1. Add 0.93 g sodium acetate and 2.32 g acetic acid to 40 ml UltraPureTM water, mix
    2. Fill up to 50 ml with UltraPureTM water

Acknowledgments

This protocol was developed with the help of the Gronert laboratory at UC Berkeley.
Funding: HHMI and NIH grants (AI075039, AI063302, EY026082), Austrian Science Fund (FWF) (the Erwin Schroedinger Fellowship J3789-B22).

Competing interests

The author declares no conflict of interest.

Ethics

The animal experiments complied with the regulatory standards of, and were approved by, the University of California Berkeley Institutional Animal Care and Use Committee.

References

  1. Rauch, I., Deets, K. A., Ji, D. X., von Moltke, J., Tenthorey, J. L., Lee, A. Y., Philip, N. H., Ayres, J. S., Brodsky, I. E., Gronert, K. and Vance, R. E. (2017). NAIP-NLRC4 inflammasomes coordinate intestinal epithelial cell expulsion with eicosanoid and IL-18 release via activation of Caspase-1 and -8. Immunity 46(4): 649-659.

简介

腹膜巨噬细胞和肠上皮细胞(IEC)中炎性体的激活导致类二十烷酸的释放。 为了评估IEC释放的类二十烷酸的量,在通过脂质谱分析或ELISA分析之前,需要从整个组织中分离脂质。 该方案描述了如何从肠组织中分离脂质用于通过PGE 2 -ELISA进行分析并归一化至组织蛋白质含量。

【背景】炎性体诱导的类花生酸释放是一个相对较新的观察结果。 目前尚不清楚除腹膜巨噬细胞和肠上皮细胞(IEC)(Rauch 等人,2017)之外的哪种细胞/组织类型可以在炎性体激活后释放前列腺素。 该方案可以适用于其他类型的组织以及测量由除肠组织中的炎性体激活之外的其他刺激诱导的类花生酸释放。

注意,该方案特别用于通过ELISA进行的类花生酸分析,已经描述了通过脂质谱法进行类花生酸分析的其他方案。

关键字:肠道, 类二十烷酸, ELISA, 前列腺素, 脂类提取

材料和试剂

  1. 培养管(Corning,Falcon,目录号:352059)
  2. 15毫升管(康宁,猎鹰,目录号:352097)
  3. 平底96孔板,透明(Corning,Costar,目录号:3370)
  4. pH试纸(GE Healthcare,Whatman,目录号:2613-991)
  5. SPE柱(C-18)(Cayman Chemical,目录号:400020)
  6. 纸巾
  7. 液氮
  8. UltraPure TM 水(Thermo Fisher Scientific,Invitrogen TM ,目录号:10977)
  9. 吲哚美辛(Sigma-Aldrich,目录号:I8280-5G)
  10. PBS(Thermo Fisher Scientific,Gibco TM ,目录号:10010)
  11. 200证明Kopec乙醇(Decon Labs,目录号:V1001)
  12. 甲醇(Fisher Scientific,目录号:A452SK)
  13. 乙酸乙酯(Sigma-Aldrich,目录号:270989)
  14. 前列腺素E 2 ELISA试剂盒(单克隆)(Cayman Chemical,目录号:514010)
  15. BCA检测试剂盒(Thermo Fisher Scientific,Pierce TM ,目录号:23227)
  16. K 2 HPO 4 (Fisher Scientific,目录号:P288)
  17. KH 2 PO 4 (Fisher Scientific,目录号:P285)
  18. DMSO(Sigma-Aldrich,目录号:D8418)
  19. EDTA(Life Technologies,目录号:15575020)
  20. 醋酸钠(Sigma-Aldrich,目录号:S2889)
  21. 乙酸(Sigma-Aldrich,目录号:A6283)
  22. 磷酸盐缓冲液(见食谱)
  23. 1 M醋酸盐缓冲液(见食谱)

设备

  1. 钳子
  2. 剪刀
  3. POLYTRON ® PT 2500 E支架分散装置(Ecoline),12毫米骨料(Kinematica)或类似产品
  4. 冰箱
  5. 离心15 ml能够冷却的反应管(Eppendorf 5810R或类似产品)
  6. 涡流
  7. 能够测量405和420 nm和562 nm之间吸光度的96孔板读数器(例如,Molecular Devices,型号:Spectramax ® M2)
  8. Reacti-Vap TM 蒸发器(Thermo Fisher Scientific,目录号:TS-18825或TS-18826)或类似产品
  9. 带压力表的氮气连接到蒸发器
  10. SPE Vacuum Manifold(Sigma-Aldrich,目录号:57250-U或类似产品)
  11. 真空捕集器(例如,Fisher Scientific,Fisherbrand TM 可重复使用的厚壁过滤器烧瓶,目录号:FB3001000)连接到中央真空系统或泵(例如,Merck,化学负荷真空压力泵,目录号:WP6122050)通过油管和橡胶塞

程序

工作流程:
class =“ke-zeroborder”bordercolor =“#000000”style =“width:800px; line-height:200%;” border =“0”cellspacing =“0”cellpadding =“2”>第1天:
动物处理和组织收获(1-4小时,取决于动物的数量)
第1天或第2天:
组织匀浆和离心(30-60分钟)

蒸发1(60-90分钟)

重悬,pH,C-18柱(30-60分钟)

蒸发2(60-90分钟)

重悬,加载ELISA(1-2小时)
第2天或第3天:
完成ELISA(2小时)

进行BCA测定(1小时)


  1. 收获肠组织并立即在液氮中快速冷冻。为了收获肠道,切开牺牲小鼠的腹膜,在胸腔下端找到胃,从胃中分离小肠,用钳子轻轻拉动,同时用剪刀从肠系膜中分离。与肛门分开并布置以获得组织的概述。确保从个体动物身上收集可比较的组织区域。
    注意:2厘米的组织很容易就足够了。小鼠在组织分离之前不需要饿死。通过在纸巾上沿选定的肠道移动镊子从而挤出大部分肠内容物就足够了,从而挤出大部分内容物。
  2. 将组织在1ml磷酸盐缓冲液/ 100mg中均化。确保组织在均质化步骤之前不会解冻,但将冷冻组织直接放入装有缓冲液的均质管中。
    注意:只有将吸头浸入带有纸巾的液体中,才能打开均质器。轻轻上下移动样品,同时均匀化,不要完全从液体中取出液体。以适当的速度和适当的时间均化以使组织完全均质化(例如,在POLYTRON上以速度26-30持续10秒 PT 2500 E)
  3. 取出30μl等分试样用于BCA测定以稍后测定蛋白质浓度,冷冻(在PBS中稀释1:10用于测定:向30μl匀浆中加入270μlPBS并混合)。
  4. 将离心机预冷至4°C。为了沉淀蛋白质,向每个管中加入100%乙醇(样品体积的四倍,例如,1ml匀浆缓冲液,使用4ml乙醇)。涡旋混匀,立即放入4°C。将样品在4℃孵育5分钟,然后在4℃,3,000 x g 离心10分钟以除去沉淀的蛋白质。将上清液转移到干净的试管中。
    注意:如果您在大量缓冲液中均质化了大量组织,则只需使用1.5 ml等分试样进行后续步骤,否则蒸发将需要很长时间。请记住在最后的计算中包含此项。将剩余的上清液保持在-20°C,直到完成隔离,可以在出现问题时用作备用。
  5. 在氮气下蒸发乙醇以完全干燥(需要60-90分钟,1.5ml) 
    1. 每16个样品使用~2 PSI压力(如果样品干燥或多或少,则增加或减少压力),相应增加。将针头向下移动到样品上一段距离,这样可以听到冒泡但没有观察到飞溅。另见图1.


      图1.氮气干燥过程中的针头定位。 应观察液体的表面波纹,但不应有任何液滴飞溅到管的两侧。

    2. 管中淡黄色的残余物是正常的。
  6. 将样品重悬于UltraPure TM 水中,并通过加入1M乙酸盐缓冲液酸化至~pH 4.0。
    注意:对于您的第一个样品,请小心添加更多的醋酸盐缓冲液并注意您需要多少,然后将相同的量添加到相同类型的样品中。对于从1.5ml蒸发的样品,重悬于300μl中,所需的量的乙酸盐缓冲液约为45μl。如果需要,样品也可以重新悬浮在更多的水中,但是这将增加步骤9中的柱时间。对于某种样品类型的第一次测试,建议有更多的样品用于pH测试。要使用非常少量的样品检查样品的pH值,请将pH测试条纵向切割至原始宽度的约1/4,并使用5μl分散滴在测试区域上。记录去除的样本量以包括在反算中。如果样品含有沉淀物,则离心以除去沉淀物。样品中的微粒物质可能会堵塞SPE盒(C-18)。
  7. 将尽可能多的C-18墨盒作为样品放在真空歧管上,关闭未使用的插座。 
  8. 将真空歧管上的真空设置为~4。用5ml甲醇冲洗,然后用5ml去离子水冲洗,制备SPE(C-18)柱。 (下降速度应为~2 /秒)。在步骤10之前,不要让SPE柱(C-18)干燥。如果样品有干燥的危险,可以使用筒下方的小阀门来调节下落速度/停止柱塞。
  9. 将样品涂在SPE柱(C-18)上,让样品完全进入包装材料。 
  10. 用5ml去离子水洗涤柱子。丢弃洗涤。让色谱柱干燥(增加真空使其完全干燥)。
  11. 用含有1%甲醇的5ml乙酸乙酯从柱中洗脱PGE 2 。确保阀门来自对此溶剂不敏感的材料(或者,如果使用其他类型的使用旋塞阀的真空歧管,则将其移除并让乙酸乙酯通过重力进入柱子,在低真空下洗脱。)。
  12. 如步骤5所述,在氮气流下将乙酸乙酯蒸发至干。非常重要的是除去所有有机溶剂,因为即使少量也会对ELISA产生不利影响(再次需要约60-90分钟)。
  13. 要重悬样品,无论初始体积如何,都要从ELISA试剂盒中加入500μlELISABuffer。该量确保试剂盒中的足够缓冲液用于样品稀释和其他试剂的制备。如果使用更多的起始材料,请在步骤14中的样品稀释中考虑这一点。涡旋。加入ELISA缓冲液后,不溶性沉淀物常常留在样品中;这不会影响测定。该样品现已准备好用于ELISA。 
  14. 根据预期的PGE 2 浓度,样品需要在ELISA缓冲液中稀释。全身炎性体激活后,建议稀释1:100。
  15. 按照Chayman chemicals协议( https:// www)中的描述执行PGE 2 ELISA .caymanchem.com / PDF文件/ 514010.pdf )。
  16. 根据试剂盒方案对组织匀浆后取出的组织匀浆的等分试样进行BCA测定。用PBS稀释组织匀浆1:10进行分析。
    注意:确保稀释磷酸盐均质缓冲液/ PBS 1/10中的标准品。

数据分析

根据ELISA方案中的描述反向计算样品PGE 2 浓度。不要忘记仅考虑一份旋转上清液的等分试样。
根据BCA测定计算样品中的蛋白质含量。
现在您可以计算样品中PGE 2 / mg总蛋白的比例。

笔记

  1. 在提取当天将样品加载到ELISA上,类二十烷酸非常不稳定,即使在-80℃下也是如此。
  2. 根据您的组织和类花生酸生产诱导,您可能必须测试用于分离的不同量的组织或ELISA中使用的不同稀释度的样品。

食谱

  1. 磷酸盐缓冲剂
    1. 储存:133g K 2 HPO 4 + 32.15g KH 2 PO 4 在1L UltraPure中 TM 水。 pH值应为7.4。 
    2. 稀释1:10,在使用前加入1 mM EDTA +10μM吲哚美辛。吲哚美辛需要在DMSO中以10μM新鲜溶解
  2. 1 M醋酸盐缓冲液
    1. 将0.93g乙酸钠和2.32g乙酸加入40ml UltraPure TM 水中,混合
    2. 用UltraPure TM 水填充至50毫升

致谢

该协议是在加州大学伯克利分校的Gronert实验室的帮助下开发的。
资金来源:HHMI和NIH拨款(AI075039,AI063302,EY026082),奥地利科学基金(FWF)(Erwin Schroedinger Fellowship J3789-B22)。

利益争夺

作者声明没有利益冲突。

伦理

动物实验符合加州大学伯克利分校机构动物护理和使用委员会的监管标准,并得到其批准。

参考

  1. Rauch,I.,Deets,KA,Ji,DX,von Moltke,J.,Tenthorey,JL,Lee,AY,Philip,NH,Ayres,JS,Brodsky,IE,Gronert,K。和Vance,RE(2017) 。 NAIP-NLRC4炎性体协调肠上皮细胞排出与类花生酸和IL-18释放通过激活Caspase- 1和-8。 Immunity 46(4):649-659。
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引用:Rauch, I. (2018). Eicosanoid Isolation from Mouse Intestinal Tissue for ELISA. Bio-protocol 8(21): e3066. DOI: 10.21769/BioProtoc.3066.
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