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
Equipment
Procedure
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
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
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
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
Hi Esther,I also think you can sacrificed mice directly without anesthesia to collect the blod for FD-measurements. Best,Jalaj
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
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
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!
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
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.
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
