from http://www.sciencedirect.com/science/article/pii/S104620230300029X

Paracellular permeability of hydrophilic tracers can be monitored with compounds that are labeled fluorescently such as dextrans or radiolabeled compounds such as tritiated mannitol or inulin, or, alternatively, proteins that can be detected with an enzymatic assay such as horseradish peroxidase (HRP). In our laboratories, we use mostly fluorescent dextrans since differently sized tracers can be analyzed by the same detection method and the use of FITC- and rhodamine-labeled dextrans allows the analysis of two different tracers in the same culture.

The experiment should be started by measuring TER for later comparison with the paracellular diffusion results. The medium should then be replaced with fresh tissue culture medium, adding 1 ml to the outer chamber and 250 μl to the inner chamber of 12-well tissue culture inserts. The cultures should then be left to equilibrate in the tissue culture incubator for at least 30 min. The fluorescent tracer is then added in 50 μl into the inner chamber, bringing the total volume to 300 μl, so that the final concentration is between 1 and 3 mg/ml. The concentration used depends on the size of the tracer since a larger fraction of a small tracer (e.g., 4-kDa FITC-dextran) will diffuse across the monolayer compared with a large tracer (e.g., 400-kDa FITC-dextran, which is excluded from the paracellular pathway if tight junctions are intact). The cultures are then incubated at 37 °C for 3 to 4 h. The filters are then removed and the diffused fluorescent tracer is measured by fluorometry (excitation, 492 nm; emission, 520 nm). If required, the actual amount of diffused dextran can be determined with the help of calibration curves established with the originally added stock solution, and kinetic experiments can be performed by removing small samples of the outer chamber medium every hour. Many investigators also quantify the amount of tracer left in the inner chamber and then determine paracellular diffusion relative to the total amount of tracer added. However, if the tracer is added as described above, variation between different samples is minimal and this step is not absolutely necessary. Experiments with other tracers such as []mannitol are done in an analogous manner but using a liquid scintillation counter to follow the tracer [17].

Neither TER nor tracer permeability measurements reflect only properties of the paracellular pathway but are a composite of the paracellular and the transcellular routes. For example, an increase in 4-kDa dextran permeability could be due to either increased paracellular diffusion and/or increased rates of fluid-phase transcytosis. Since the paracellular route exhibits a much stronger size selectivity than transcytosis, a first important experiment will be to determine permeability of large tracers such as 40- and/or 400-kDa FITC-dextran. Additionally, fluid-phase transcytosis can be directly measured by allowing cells to internalize high concentrations of HRP for 10 min (3–10 mg/ml [17]). The monolayers are then extensively washed to remove all external HRP and are then reincubated at 37 °C for 1 to 2 h to allow cells to transport maximal amounts of HRP to the opposite site. The amount of transcytosed HRP can then be measured enzymatically [60].

Like tracer diffusion, TER is a composite of transcellular and paracellular resistance. In most low-resistance epithelia such as MDCK II cells, the electrical resistance of the paracellular route is much lower than the transcellular resistance [61], [62] and [63]. Since the two pathways are arranged in parallel, 1/TER=(1/Rtranscellular)+(1/Rparacellular); hence, the measured TER essentially reflects paracellular resistance. Moreover, the paracellular resistance is the sum of the junctional resistance and the resistance along the paracellular space. Therefore, a collapse of the paracellular space can cause a non-ion-selective increase in TER (as well as a decrease in paracellular permeability). Hence, a careful ultrastructural morphological analysis can be important to understand changes in TER and tracer permeability.

See the ref below. They used similar to yours set up and Coco-2 cells. Hope this will be useful.

Pharmaceutical Research

August 1993, Volume 10, Issue 8, pp 1123-1129

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

Per Artursson,

Anna-Lena Ungell,

Jan-Erik Löfroth

FITC-conjugated dextrans can be bought in different sizes, so flux from apical application to basal compartment of transwell can be measured on a fluorescent plate reader. Easy assay to do - hope it helps!

from http://www.sciencedirect.com/science/article/pii/S104620230300029X

Paracellular permeability of hydrophilic tracers can be monitored with compounds that are labeled fluorescently such as dextrans or radiolabeled compounds such as tritiated mannitol or inulin, or, alternatively, proteins that can be detected with an enzymatic assay such as horseradish peroxidase (HRP). In our laboratories, we use mostly fluorescent dextrans since differently sized tracers can be analyzed by the same detection method and the use of FITC- and rhodamine-labeled dextrans allows the analysis of two different tracers in the same culture.

The experiment should be started by measuring TER for later comparison with the paracellular diffusion results. The medium should then be replaced with fresh tissue culture medium, adding 1 ml to the outer chamber and 250 μl to the inner chamber of 12-well tissue culture inserts. The cultures should then be left to equilibrate in the tissue culture incubator for at least 30 min. The fluorescent tracer is then added in 50 μl into the inner chamber, bringing the total volume to 300 μl, so that the final concentration is between 1 and 3 mg/ml. The concentration used depends on the size of the tracer since a larger fraction of a small tracer (e.g., 4-kDa FITC-dextran) will diffuse across the monolayer compared with a large tracer (e.g., 400-kDa FITC-dextran, which is excluded from the paracellular pathway if tight junctions are intact). The cultures are then incubated at 37 °C for 3 to 4 h. The filters are then removed and the diffused fluorescent tracer is measured by fluorometry (excitation, 492 nm; emission, 520 nm). If required, the actual amount of diffused dextran can be determined with the help of calibration curves established with the originally added stock solution, and kinetic experiments can be performed by removing small samples of the outer chamber medium every hour. Many investigators also quantify the amount of tracer left in the inner chamber and then determine paracellular diffusion relative to the total amount of tracer added. However, if the tracer is added as described above, variation between different samples is minimal and this step is not absolutely necessary. Experiments with other tracers such as []mannitol are done in an analogous manner but using a liquid scintillation counter to follow the tracer [17].

Neither TER nor tracer permeability measurements reflect only properties of the paracellular pathway but are a composite of the paracellular and the transcellular routes. For example, an increase in 4-kDa dextran permeability could be due to either increased paracellular diffusion and/or increased rates of fluid-phase transcytosis. Since the paracellular route exhibits a much stronger size selectivity than transcytosis, a first important experiment will be to determine permeability of large tracers such as 40- and/or 400-kDa FITC-dextran. Additionally, fluid-phase transcytosis can be directly measured by allowing cells to internalize high concentrations of HRP for 10 min (3–10 mg/ml [17]). The monolayers are then extensively washed to remove all external HRP and are then reincubated at 37 °C for 1 to 2 h to allow cells to transport maximal amounts of HRP to the opposite site. The amount of transcytosed HRP can then be measured enzymatically [60].

Like tracer diffusion, TER is a composite of transcellular and paracellular resistance. In most low-resistance epithelia such as MDCK II cells, the electrical resistance of the paracellular route is much lower than the transcellular resistance [61], [62] and [63]. Since the two pathways are arranged in parallel, 1/TER=(1/Rtranscellular)+(1/Rparacellular); hence, the measured TER essentially reflects paracellular resistance. Moreover, the paracellular resistance is the sum of the junctional resistance and the resistance along the paracellular space. Therefore, a collapse of the paracellular space can cause a non-ion-selective increase in TER (as well as a decrease in paracellular permeability). Hence, a careful ultrastructural morphological analysis can be important to understand changes in TER and tracer permeability.

As mentioned by Karen Wright, graded series of fluorescently labeled dextrans are frequently used. Otherwise, mannitol, inulin or graded PEG polymers can be used. However, be aware that TEER and permeability are different aspects. Please also see Anderson JM and Van Itallie CM, Cold Spring Harbor Perspectives in Biology 2009,; 1:a002584.

Dear Marcia,

Thank you very much for contacting me.

In my experience one of the more accepted method to test the Paracellular permeability is to use a chemical compound labelled with a tracer that is not carried by a healthy cell monolayer.

For the Human intestinal cells the best choice is to use a C14-labelled-Mannitol loaded into the upper chamber of a transwell insert. Mannitol, in fact, is a sugar that is not naturally carried by intestinal cells. After 1 h from the load you can read any trace of C14 in the lower chamber and estimate the P.app or Apparent Permeability of your monolayer. It is important to label the Carbon bone of the molecule since if you label the Hydrogen instead there is a high risk that the H3 will exchange with the H2 of the water around and you will read an high content of radioactive in the lower chamber because of the labelled water carried by the aquaporins distributed in the cell membrane.

For your system I don't know which is the best not naturally carried compound you can label but I am sure there is something that can suit your needs. Otherwise you can just try with mannitol and see what happens.

I hope this will help you a bit and I wish you very good luck with your experiments.

Manuela

Apart from all the good suggestions you have received i recommend you to label one of the tight-junction proteins, for example, occludin (the most common) with and antibody conjugated with some fluorescent dye. That way you can verify if the paracellular way is really compromised or not.

In case you need. I have a couple of papers where i do the same after TEER measurements with Caco-2 cells.

You can check them out here below:

http://onlinelibrary.wiley.com/doi/10.1111/febs.12390/abstract

http://www.mdpi.com/2072-6651/6/1/211

Let me know if you need further information.

I measured paracellular permeability of endothelial cell monolayer grown in an insert cell culture device with two methods : ZO-1 (Zonula Occludens 1 protein) staining of tight junctions complexes ; and FITC-Dextran 4400 Da permeability.

II suggest you to look my last article recently published in Gene (2013) concerning the quantification of the permeability of pulmonary or muscle vascular endothelial barriers, where you will find a complete description of the materials and methods of these techniques, references and results.

The pdf can be downloaded freely on my Researchgate page.

Do not hesitate if you have any questions.

Sincerly yours.

Just keep in mind the size of the tracer you will use in the paracellular permeability assays that people have already described in detail. TEER measurements depend on the electrical current that is carried by ions in the solution and therefore will reflect the permeability of the monolayer to small molecules. Dextrans larger than 5 kDa will give you information about the diffusional permeability of larger molecules that may or may not be relevant for your experimental design - in our lab, for example, we calculate endothelial monolayer permeability to FITC-Dextran 70 kDa, which is a good mimic of albumin, the most abundant plasma protein. Stay away from small molecules that are known to be transported transcellularly in the specific epithelium you're working on.

Good luck!

Use vital stains as tryban blue

Hi Marcia

I actually measured plasma membrane permeability (permeability coefficient), but this is in a plant cells with a cell wall. The technique is developed by EJ Stadelmann. I do not know whether it can work for epithelial cells. You can find more detail in Stadelmann and Lee-Stadelmann 1989. Passive permeability. Method. Enzymol. 174: 246-266., and also in Mansour et al 1993. Physiol. Plant. 88: 141-148.

Good luck!

Thank you very much for all the suggestions! I will take a look on then and choose the best for my assay!

Dear Maria,

If you have the possibility to use electron microscopy techniques, you could also test Lanthanum tracer: Below I have suggested couple of artcles concerning my experience of the tracer. I also add an attachment desciribing how we developed a method for using Lanthanum sutiable for our partuclular needs.

Kind regards

Harriet

CFTR and tight junctions in cultured bronchial epithelial cells

Harriet E. Nilsson, Anca Dragomir, Lucia Lazorova, Marie Johannesson, Godfried M. Roomans

Experimental and Molecular Pathology 88 (2010) 118–127

Effects of hyperosmotic stress on cultured airway epithelial cells

Harriet Nilsson & Anca Dragomir & Anders Ahlander &

Marie Johannesson & Godfried M. Roomans

Cell Tissue Res (2007) 330:257–269

I am using FITC dextran to evaluate parcellular permeability on endothelial cells monolayer. Easy to use. You can even add a positive control such as DMSO that will disrupt the junctions to have the maximal value of permeability or an empty well. The other thing I do as suggested above is to use antibodies directed against proteins involved in junctions such as occludin. Good luck on your experiment.

Hi. All the suggestions above seem to be good and you have to decide which gives you the best output for the questions you are going to ask (depending on the MW of the used tracer). A very precise method, if you have a very tight epithelial barrier, is radiolabeled sucrose, but I am not sure if it is worth the effort. We also worked with different sized fluorescence-labeled dextranes.

Another point I want to mention is that, if you have a very sensitive cell culture and you want to maintain it with the polarized cell surfaces, you should be very careful with the exchange of the media for the permeability assays and maybe only replace a certain part of the media. Otherwise you may interfere with the cell polarity and increase the permeabilty of the cell layer just by your handling!

Everyone here is making very good suggestions. The only thing I would add is that paracellular permeability is not a cell characteristic per se. Paracellular permeability is defined with reference to a particular solute. This is very evident in renal epithelial cells, where there can be good paracellular permeability to divalent cations such as calcium, yet essentially no paracellular permeability to other divalent cations such as magnesium.

I'd add the technique used by Dr. J Anderson's group, i.e., the permeability assay using polyethyleneglycols (PEG) of various sizes. Also, I also would recommend the 'equivalent pore analysis' using the observed permeability characteristics It is rather archaic and demanding analysis, but it could shed some light in terms of comparison purposes. For example, one could determine if the experimental maneuver led to tighten up/loosen up/no change in tight junctional properties and/or to simply compare the barrier properties under test with those of other barriers. Highly recommended to read Dr. Anderson's papers listed below [especially the 2011 paper]:

Measuring size-dependent permeability of the tight junction using PEG profiling.

Van Itallie CM, Anderson JM.

Methods Mol Biol. 2011;762:1-11.

doi: 10.1007/978-1-61779-185-7_1.

PMID: 21717345

The density of small tight junction pores varies among cell types and is increased by expression of claudin-2.

Van Itallie CM, Holmes J, Bridges A, Gookin JL, Coccaro MR, Proctor W, Colegio OR, Anderson JM.

J Cell Sci. 2008 Feb 1;121(Pt 3):298-305.

doi: 10.1242/jcs.021485.

PMID: 18198187

We use TEER, fitC-dextran, and Lucifer yellow in our lab to measure monolayer permeability in caco2 cells. We also use fluorescent microscopic colocalization of ZO-1/occludin and F-actin to visualize tight junctions in epithelial intestinal sections. All are pretty straight forward