搜索

Analysis of Intestinal Permeability in Mice
小鼠肠道通透性分析   

评审
匿名评审
下载 PDF 引用 收藏 3 提问与回复 分享您的反馈 Cited by
Ask the Authors

本文章节

Abstract

The intestinal epithelial layer serves as a barrier against pathogens and ingested toxins, which are present in the lumen of the intestine. The importance of the intestinal epithelial barrier is emphasized by the alterations in paracellular permeability and tight junction functions observed in inflammatory bowel disease (IBD) and colon cancer.

Keywords: epithelial barrier(上皮屏障), inflammation(炎症), tumor(肿瘤), FITC-dextran(FITC-葡聚糖)

Materials and Reagents

  1. 8-10-week old mice (in-house bred mice mostly C57BL/6 background; both male and female)
  2. Fluorescein isothiocyanate conjugated dextran (FITC-dextran) (Sigma-Aldrich, catalog number: FD4 )
  3. Sterile 1x phosphate-buffered saline (PBS) (pH 7.4)
  4. Anesthesia (isoflurane) (ESTEVE, catalog number: 103287025 )

Equipment

  1. BD Microtainer SST tubes (BD Biosciences, catalog number: 365968 )
  2. 1 ml syringes (ENFA, catalog number: JS1 )
  3. Autoclaved oral gavage needles (22 Gauge /25 mm long) (Fine Science Tools, catalog number: 18061-22 )
  4. Spectrophotofluorometer (BioTek Instruments, catalog number: FLx800 )
  5. 96-well microplates (flat bottom) (Corning, catalog number: 3650 )
  6. Dissection equipment (forceps, tweezers, scissors)
  7. Balance  
  8. Table top anesthesia machine (Parkland Scientific, catalog number: V3000PK )

Procedure

  1. On day 1, in the evening remove water bottles from the cages to water starve the mice overnight.
  2. On day 2, in the morning weight the mice.
  3. Dissolve FITC-dextran in PBS at a concentration of 100 mg/ml and administer FITC-dextran to each mouse (44 mg/100 g body weight) by oral gavage with a needle attached to a 1 ml syringe. The procedure for oral gavage is described elsewhere (Bertola et al., 2013). A gap of 30 min between each mouse is recommended for the FITC-dextran oral gavage (see Notes).
  4. After 4 h, anesthetize the mice by isoflurane inhalation and collect the blood using a 1 ml syringe with 25 G needle by cardiac puncture, then kill the mice by cervical dislocation. Isoflurane is delivered at a concentration of 4% in oxygen using a precision vaporizer for the initial induction and then is maintained at 3% during the blood collection. Typically, we collect 300-400 μl of blood in order to get enough serum for the next step.
  5. Immediately, transfer blood to a Microtainer SST tube, mix by inverting the tube 3-4 times and store at 4 °C in in the dark. Once blood has been collected from all the mice, SST tubes are processed to separate the serum following the manufacturer’s instruction.
  6. Dilute the serum with an equal volume of PBS.
  7. Add 100 μl of diluted serum to a 96-well microplate in duplicate.
  8. Determine the concentration of FITC in serum by spectrophoto fluorometry with an excitation of 485 nm (20 nm band width) and an emission wavelength of 528 nm (20 nm band width) using as standard serially diluted FITC-dextran (0, 125, 250, 500, 1,000, 2,000, 4,000, 6,000, 8,000 ng/ml) (Figure 1). Serum from mice not administered with FITC-dextran is used to determine the background.

Representative data



Figure 1. Standard curve for the intestinal permeability assay showing linearity over the range of concentrations tested


Figure 2. Intestinal permeability measured by determining the concentration of FITC-dextran in the serum of WT and p38α-ΔIEC mice. Data are means ± SEM (n = 4). *p < 0.05

Notes

  1. The administration of FITC-dextran with a gap of 30 min between animals allows for anesthetization and collection of blood by cardiac puncture from each mouse while maintaining the 4 h interval between FITC-dextran administration and blood collection.
  2. In different experiments, we observed FITC concentration ranges of 500-800 ng/ml in the serum of WT mice. The mean concentration of FITC in the serum of WT mice administered with FITC-dextran, without any further treatment, should be considered normal. In our case (Gupta et al., 2014), we found that mice with deletion of p38α in intestinal epithelial cells (p38α-ΔIEC) show higher concentration of FITC-dextran in the serum and therefore concluded that intestinal permeability was enhanced in these mice compared to WT mice (Figure 2).

Acknowledgments

Our work is supported by the Fundación BBVA and by grants from the Spanish MICINN (BFU2010-17850) and the European Commission FP7 (INFLA-CARE 223151 and ERC 294665). This protocol is a modification of the protocol published by Calon et al. (2007).

References

  1. Bertola, A., Mathews, S., Ki, S. H., Wang, H. and Gao, B. (2013). Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc 8(3): 627-637.
  2. Calon, A., Gross, I., Lhermitte, B., Martin, E., Beck, F., Duclos, B., Kedinger, M., Duluc, I., Domon-Dell, C. and Freund, J. N. (2007). Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation. Gut 56(12): 1688-1695.
  3. Gupta, J., del Barco Barrantes, I., Igea, A., Sakellariou, S., Pateras, I. S., Gorgoulis, V. G. and Nebreda, A. R. (2014). Dual function of p38alpha MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival. Cancer Cell 25(4): 484-500.

简介

肠上皮层用作针对存在于肠腔中的病原体和摄入的毒素的屏障。 肠上皮屏障的重要性通过在炎症性肠病(IBD)和结肠癌中观察到的细胞旁通透性和紧密连接功能的改变来强调。

关键字:上皮屏障, 炎症, 肿瘤, FITC-葡聚糖

材料和试剂

  1. 8-10周龄小鼠(自体繁殖的小鼠大多为C57BL/6背景;雄性和雌性)
  2. 荧光素异硫氰酸酯结合的葡聚糖(FITC-葡聚糖)(Sigma-Aldrich,目录号:FD4)
  3. 无菌1×磷酸盐缓冲盐水(PBS)(pH 7.4)
  4. 麻醉(异氟烷)(ESTEVE,目录号:103287025)

设备

  1. BD Microtainer SST管(BD Biosciences,目录号:365968)
  2. 1ml注射器(ENFA,目录号:JS1)
  3. 高压灭菌的口服管饲针(22Gauge/25mm长)(Fine Science Tools,目录号:18061-22)
  4. 分光光度计(BioTek Instruments,目录号:FLx800)
  5. 96孔微孔板(平底)(Corning,目录号:3650)
  6. 解剖设备(镊子,镊子,剪刀)
  7. 余额
  8. 台式麻醉机(Parkland Scientific,目录号:V3000PK)

程序

  1. 在第1天,晚上从笼子中取出水瓶,使水饥饿过夜。
  2. 在第2天,在早晨的体重小鼠
  3. 以100mg/ml的浓度溶解于PBS中的FITC-葡聚糖,并通过用连接至1ml注射器的针口服管饲法给每只小鼠施用FITC-葡聚糖(44mg/100g体重)。口服管饲的方法在别处描述(Bertola等人,2013)。对于FITC-葡聚糖口服管饲法,推荐每只小鼠之间有30分钟的间隙(见注释)
  4. 4小时后,通过异氟醚吸入麻醉小鼠,并使用1毫升注射器用25 G针通过心脏穿刺收集血液,然后通过颈脱位法杀死小鼠。使用精确蒸发器在氧气中以4%的浓度输送异氟烷用于初始诱导,然后在血液收集期间维持在3%。通常,我们收集300-400μl的血液,以获得足够的血清进行下一步。
  5. 立即,将血液转移到Microtainer SST管,混合通过翻转管3-4次,并存储在4°C在黑暗中。一旦从所有小鼠收集血液,就按照制造商的说明处理SST管以分离血清
  6. 用等体积的PBS稀释血清。
  7. 将100μl稀释的血清加入96孔微孔板中一式两份
  8. 使用作为标准系列稀释的FITC-葡聚糖(0,125,250和250nm),使用485nm(20nm带宽)的激发和528nm(20nm带宽)的发射波长通过分光光度荧光法测定血清中FITC的浓度。 500,1,000,2,000,4,000,6,000,8,000ng/ml)(图1)。来自未施用FITC-葡聚糖的小鼠的血清用于测定背景

代表数据



图1.肠道渗透性测定的标准曲线,显示在测试浓度范围内的线性度


图2.通过测定WT和p38α-Δ IEC 小鼠血清中FITC-葡聚糖的浓度测量的肠通透性。 ±SEM(n = 4)。 * p < 0.05

笔记

  1. 在动物之间间隔30分钟的FITC-葡聚糖的施用允许麻醉和通过从每只小鼠心脏穿刺收集血液,同时保持FITC-葡聚糖施用和血液收集之间的4小时间隔。
  2. 在不同的实验中,我们在WT小鼠的血清中观察到500-800ng/ml的FITC浓度范围。在施用FITC-葡聚糖的WT小鼠的血清中的FITC的平均浓度应当被认为是正常的,而没有任何进一步的治疗。在我们的情况下(Gupta等人,2014),我们发现在肠上皮细胞中缺失p38α的小鼠(p38α-Δ IEC )显示更高浓度的FITC-葡聚糖,因此得出结论,与WT小鼠相比,这些小鼠的肠通透性增强(图2)。

致谢

我们的工作得到BBVA基金和西班牙MICINN(BFU2010-17850)和欧盟委员会FP7(INFLA-CARE 223151和ERC 294665)的资助。该协议是由Calon等人(2007)发布的协议的修改。

参考文献

  1. Bertola,A.,Mathews,S.,Ki,S. H.,Wang,H.和Gao,B.(2013)。 慢性和无症状乙醇喂养的小鼠模型(NIAAA模型)。 Nat Protoc 8(3):627-637。
  2. Calon,A.,Gross,I.,Lhermitte,B.,Martin,E.,Beck,F.,Duclos,B.,Kedinger,M.,Duluc,I.,Domon-Dell,C.and Freund,JN (2007)。 Cdx1和Cdx2同源盒基因在小鼠肠炎模型中的不同作用。 Gut 56(12):1688-1695。
  3. Gupta,J.,del Barco Barrantes,I.,Igea,A.,Sakellariou,S.,Pateras,I.S.,Gorgoulis,V.G.and Nebreda,A.R。(2014)。 p38alpha MAPK在结肠癌中的双重功能:抑制结肠炎相关的肿瘤起始,但对癌细胞的需求生存。癌细胞 25(4):484-500。
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gupta, J. and Nebreda, A. R. (2014). Analysis of Intestinal Permeability in Mice. Bio-protocol 4(22): e1289. DOI: 10.21769/BioProtoc.1289.
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。

Hai Phung
Tohoku univ. Graduate school of Medicine
Dear Dr. Gupta,

Thank for your protocol.

I have 2 questions:
(1) serial serum dilution: to make the standard curve, could I dilute FITC-Dextran in WT-mice serum to 10, 20, 40, 80 ng/ml. Could you suggest the lowest concentration that could be still detectable?
(2) Could I use 50 mcl instead of 100mcl 2x diluted serum as the protocol mentions?
Thank you,

Hai.
10/5/2016 8:15:39 PM Reply
Jalaj Gupta
Institute for Research in Biomedicine (IRB Barcelona), Spain

Dear Hai Phung,
Thank you for your interest in our protocol.
1) In my experience, I could detect minimum of 400ng/ml in the mouse serum. In theory you can dilute the FITC-Dextran in WT mice serum but you should include standards up to 500-600ng/ml (eg: 10, 50, 100, 200, 400, 600 ng/ml). We used 125ng/ml as lowest standard, which was detected easily.
2) We always used 100ul diluted serum and could detect the FITC-dextran quite nicely but I think you can still dilute the serum to detect FITC-dextran and then multiply by dilution factor in order to get final concentrations.
I hope this will help you,
best,
Jalaj

10/10/2016 1:51:30 AM


LI YEQIONG
NANJING UNIVERSITY
Dear Dr. Gupta,

I have a question about your protocol. If I want to analysis the gut permeability of wild type mice and IL6-/- mice, should I use two standard curves respectively to determine the concentration of FITC in serum? For example, for IL6-/- group, should I use serum from untreated IL6-/- mice to determine the backgroud? And for WT group, should I use serum from untreated WT mice to determine the backgroud?

Thanks a lot for your help.
3/29/2016 1:39:11 AM Reply
Angel Nebreda
Institute for Research in Biomedicine (IRB Barcelona), Spain

Dear LI,
Thank you for your interest in our protocol. You can use only one standard curve and serum from one of the untreated WT mice of same genetic background (better littermate) to determine the concentration of FITC-dextran in the serum samples from both WT and IL6 KO mice.
I hope this will help you.
Good Luck,
Best,
Jalaj Gupta

3/29/2016 4:35:04 AM


LI YEQIONG
NANJING UNIVERSITY

Dear Dr. Gupta,
Thanks for your reply. I have some more questions.
1. How long can I keep the serum samples at 4 °C until the spectrophoto determination?
2.Can I store the serum samples at -20°C or -80°C until I collect enough samples for spectrophoto determination? Will it change the activity of fluorescein isothiocyanate?
3. How long can the FITC-dextran clear out of body compeletely?After oral gavage of FITC-dextran for 4h, can I collect serum sample but not kill the mice, keep the mice for somedays and gavage FITC-dextran again and collect serum sample?
Thank you a lot for your reply.

Best wishes!

4/6/2016 3:24:46 AM


Angel Nebreda
Institute for Research in Biomedicine (IRB Barcelona), Spain

Dear LI,
1. We kept samples at 4 ºC for maximum of 2h after taking it out from mice and immediately analyzed for FITC determination by spectrophotometer.
2. As I said we always used fresh serum to analyze FITC concentration in serums but in theory if you protect from light and samples are nicely and similarly stored at -20ºC, you should be able to detect FITC with minimum loss of its Fluorescence.
3. I can’t exactly answer your question about how long it will take to clear out FITC-dextran from blood. This you should check. You do not need to kill mice after 4h if you are able to take around 250ul blood to get atleast 100 ul serum.
best,
Jalaj

4/7/2016 4:37:21 AM


LI YEQIONG
NANJING UNIVERSITY

Dear Dr. Gupta,

Thanks a lot for your reply. I'm sorry to bore you again. I have 2 questiones.

1. Why you remove the water bottle for 5-6 h or overnight before the FITC-dextran oral gavage? In some papers there are no mention of water starvation.

2. What kind of 96-well microplate are you used? Black or white? Do we need transparent bottom of the microplate?

Best wishes.

4/7/2016 7:43:47 PM


Busra Yagabasan
ETH Zurich
Dear Dr. Gupta,

I plan to use your protocol for my experiment. But I have 2 questions. I would be grateful if you can help me about them.

The first question is: Do you starve the mice including also food overnight?

Second question is: After the oral gavage, when you are waiting for 4 hours, do you put the water bottles back? I guess you need to put it, otherwise the blood will be so sticky and the serum in that amount (approximately 100 ul) cannot be collected.

Thanks a lot for your help,
2/24/2016 12:50:22 AM Reply
Angel Nebreda
Institute for Research in Biomedicine (IRB Barcelona), Spain

Dear Bursa,

Thank you very much for your interest in our protocol.
Yes, we only water starve the mice as also mentioned in the protocol. In my recent experience, 5-6 h of water starvation also reduces variability between mice.
Regarding your second query, during the 4h waiting time we put the water bottle back.
I hope this information will help you to design your experiment.

Best,
Jalaj Gupta

2/25/2016 1:22:50 AM


LI YEQIONG
NANJING UNIVERSITY

3/29/2016 1:38:06 AM