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Analysis of Intestinal Permeability in Mice    

Jalaj GuptaJalaj Gupta Angel R. NebredaAngel R. Nebreda
DOI:   10.21769/BioProtoc.1289
Published:   Vol 4, Iss 22, November 20, 2014
Cell Biology > Tissue analysis > Tissue staining
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In this protocol

Original research article

A brief version of this protocol appeared in:
Apr 2014

 

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, inflammation, tumor, FITC-dextran

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.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Gupta, J. and Nebreda, A. R. (2014). Analysis of Intestinal Permeability in Mice. Bio-protocol 4(22): e1289. DOI: 10.21769/BioProtoc.1289.
Q&A

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Esther Mezhibovsky
Esther Mezhibovsky
Rutgers
Dear Dr. Gupta,

Do you know if FITC-Dextran will interfere with immunofluorescence staining performed on tissues after the assay?
8/21/2018 9:44:10 AM Reply
Feng Wei
Feng Wei
Fudan University
Dear Dr. Gupta, Thanks for your protocol. I have 2 questiones. 1. What kind of 96-well microplate are you used? Black or white? 2. Does this experiment affect ELISA for serum inflammatory factors? Best wishes.
8/3/2018 12:36:14 PM Reply
qiulan lv
qiulan lv
骨研所
Thank for your protocol. I have some quenstion： 1. the aims of remove the water overnight to starve the mice ? 2. there are effects on weights between control and treated mice if i remove the water , so is it necessary to remove the water? Thanks for your answers.
6/9/2018 5:06:48 PM Reply
Jalaj Gupta
Jalaj Gupta
Stem Cell Research Center (SCRC), Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow

Hi,
1) In our experience when we removed water (overnight or 4 to 6h before FITC-dextran), we see less variability in FITC-dextran concentration in bood in WT mice; probably because of even absorption of FD4.
2) we never did FD4 measurements in treated mice. We used untreated WT or KO mice and weight loss due to water removal for overnight or 4-6h was very minimal. I would suggest to remove water (you can reduce the time to 2-3h) only if treated mice are healthy.
I hope this will help you,
Good luck,
Jalaj

6/16/2018 2:09:27 AM

Esther Mezhibovsky
Esther Mezhibovsky
Rutgers
Hello,
May this protocol be followed without anesthetizing the mice, and rather sacrificing prior to blood draw?

Thanks
5/31/2018 9:51:46 AM Reply
Jalaj Gupta
Jalaj Gupta
Stem Cell Research Center (SCRC), Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow

Hi Esther,
I also think you can sacrificed mice directly without anesthesia to collect the blod for FD-measurements.
Best,
Jalaj

6/16/2018 2:14:23 AM

Hai Phung
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
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
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 R. Nebreda
Angel R. 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
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 R. Nebreda
Angel R. 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
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
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 R. Nebreda
Angel R. 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
LI YEQIONG
NANJING UNIVERSITY

3/29/2016 1:38:06 AM

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