The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.
Applying Lucifer Yellow to the media of Apical-out enteroids provides a simple method for quantitative and qualitative analysis of all three major paracellular intestinal permeability pathways in a 3D model. It is a relatively straightforward technique that allows us to assess permeability using an enteroid model. We are working with this technique in our studies of necrotizing enterocolitis.
However, this technique can be useful in studying any human intestinal disease, utilizing an enteroid model. Demonstrating the procedures today will be our Post-Doctoral Research Scholars, Dr.Tyler Leiva, and Dr.Alena Golubkova, as well as Camille Schlegel, the Research Assistant Supervisor from our laboratory. To begin, grow the Androids embedded in BMM for seven to 10 days with 50%LWRN conditioned media for the apical-out or AO enteroid generation.
Remove the media from around the BMM domes and add 500 microliters of cell recovery solution, or CRS to one well. Scrape the dome, pipette it up and down several times to homogenize the CRS with the enteroids embedded in BMM domes, and add this solution to the next well. Again, scrape the dome and pipette it up and down several times before transferring the solution to a microcentrifuge tube.
Add 500 microliters of CRS to the second well, pipette up and down to mix it well and transfer it to the first well. From the first well, pool the CRS enteroid BMM solution into the same 1.5 milliliters microcentrifuge tube and repeat this pooling for the remaining wells until the contents of all the wells are in CRS. Place the microcentrifuge tubes containing pooled CRS solution on a rotator for one hour at four degrees Celsius.
Then centrifuge the microcentrifuge tube at 200 X g for three minutes at four degrees Celsius before removing the supernatant, and re-suspending the enteroid pellet in one milliliter of 50%LWRN conditioned media. Gently transfer 500 microliters of the re-suspended enteroid or media solution to each well of the ultra-low attachment 24 well tissue culture plate. Incubate the plate at 37 degrees Celsius with 5%CO2, for three days prior to experimentation.
Ensure that AO enteroids are suspended for at least 72 hours before use. Gently transfer all the enteroids suspended in media from each well into a microcentrifuge tube. Centrifuge at 300 X g for three minutes at room temperature and remove the supernatant.
Add 10 microliters of five milligrams per milliliter of lipopolysaccharide, or LPS to 500 microliters of 50%LWRN conditioned media per well to prepare the master mix, and re-suspend the AO enteroid pellet from the treatment group into 500 microliters of LPS plus 50%LWRN conditioned media. Pipette up and down to thoroughly mix. Aliquot 500 microliters of suspension into each well of a 24 well ultra low attachment plate.
Similarly re-suspend the AO enteroids from the untreated control group in 500 microliters of 50%LWRN conditioned media per well and aliquot 500 microliters of this suspension into each well of a separate 24 well ultra-low attachment plate. Induce hypoxia in the LPS treated AO enteroids using a modular incubator chamber with 1%oxygen, 5%carbon dioxide, and 94%nitrogen, and incubate the enteroids with hypoxia and LPS for 24 hours. Place the untreated and LPS plus hypoxia, treated cultures in a 5%carbon dioxide incubator for 24 hours.
To measure the paracellular permeability using Lucifer Yellow or LY, gently transfer the enteroid or media suspension from each well into a microcentrifuge tube. Warm Dulbecco's phosphate buffered saline or DPBS, and 50%LWRN conditioned media in a 37 degrees Celsius water bath. Centrifuge the suspension at 300 X g for three minutes at room temperature.
Remove the media, and wash the enteroid pellet with 500 microliters of warm DPBS before centrifugation at 300 X g for three minutes. Remove the supernatant with a micro pipette and repeat the DPBS wash step one more time. After the DPBS washes, add 450 microliters of warm 50%LWRN conditioned media, and add the suspension to a 24 well plate.
Then add 50 microliters of 2.5 milligrams per milliliter LY to each well. Swirl the plate gently to mix, and place it in a 5%carbon dioxide incubator for two hours. Gently pipette the enteroid suspension from each well into a microcentrifuge tube.
Centrifuge it and remove the supernatant leaving the enteroid pellet behind. Wash the pellet using 500 microliters of warm DPBS and centrifuge it. Remove the supernatant leaving the pellet behind.
Repeat three washes of the DPBS before re-suspending the pellet with 1000 microliters of cold DPBS, and dissociate the enteroids by vigorous mixing using a pipette. For the Lucifer Yellow standard curve, prepare the standards diluted in DPBS as described in the text. Pipette 150 microliters of each sample per well in triplicate on a 96 well plate.
Measure the fluorescence at an excitation peak of 428 nanometers and an emission peak of 536 nanometers on a fluorescence plate reader. Calculate the concentrations using statistical software and a four parameter curve interpolating the standard curve. Immunofluorescent staining of the enteroids suspended in 50%LWRN media for 72 hours showed Zo-1 on the outer apical surface of the enteroid, while beta catenin is seen on the basolateral surface.
It indicated the expected polarity reversal of enteroids confirming an AO conformation of enteroids. Basolateral out, or BO enteroids treated with LPS and hypoxia showed significantly increased permeability which was demonstrated in a Lucifer Yellow, or LY assay by the increased uptake of LY dye into the enteroid lumen, compared to untreated controls. Lucifer Yellow assay also demonstrated the increased uptake of LY in the lumen of treated AO enteroids compared to untreated controls.
It confirmed the increased permeability of AO enteroids treated with LPS and hypoxia, compared to the untreated controls. It's important to thoroughly wash the enteroid pellets to ensure that all the extraluminal LY is removed. It allows the accurate quantification of the remaining intraluminal LY in calculating the permeability.
For qualitative analysis, enteroids can be visualized via fluorescent microscopy for LY uptake into the lumen. Following LY incubation and subsequent wash steps before dissociating from Microplate reading.
