## 2 Materials and Methods
All indicated time points refer to time beyond 24 h (i.e., for the 1 h time point, cells were prepared 25 h after seeding; for 6 h time point, 30 h after seeding; for 24 h time point, 48 h after seeding; for 48 h time point, 72 h after seeding). Monolayer Integrity. Transepithelial/transendothelial electrical resistance (TEER) is an indicator of the modulation of the passage of substances through a membrane, (i.e., decreased integrity is related to increased permeability) [42]. Data collected was used to monitor cell culture confluence, monolayer formation, and epithelial barrier function [43]. The electrical resistance of a cellular monolayer, measured in ohms, quantitatively measured the barrier integrity and the TEER measurements on the triculture model may be interpreted as a \"simulated\" intestinal barrier integrity [35]. Caco-2, HT29-MTX, and Raji B cells were seeded individually at 13,000 cells/cm\({}^{2}\) density on Transwell\({}^{\circledR}\) inserts (membrane area 1.1 cm\({}^{2}\), pore size 0.4 \(\upmu\)m, Corning\({}^{\circledR}\), Corning, NY, USA) in triplicate. The triple cell culture was seeded in triplicate as follows: Caco-2 cells at 6000 cells/cm\({}^{2}\) density in the Transwell\({}^{\circledR}\) insert, Raji B cells at 60,000 cells/cm\({}^{2}\) density in the Transwell\({}^{\circledR}\), and HT29-MTX cells at 1000 cells/cm\({}^{2}\) density were seeded on top of the Caco-2 cells in the Transwell\({}^{\circledR}\) insert. TEER was measured twice daily for seven days using an epithelial volothmmeter (EVOM) with \"chopstick\" electrodes (World Precision Instruments, LLC, Sarasota, FL, USA). The monocultures were measured after confluency and up to 7 days, while the triple co-culture was measured after differentiation was reached (approximately 5 days), then measured for resistance. Resultant data was an averaged triplicate with each value measured from a different point in the same well. Oxidative Stress. ROS-Glo\({}^{\circledR}\) H\({}_{2}\)O\({}_{2}\) luminescent assay (Promega, Madison, WI, USA) was utilized to assess oxidative stress in cells at varying seeding densities (4000; 7000; 10,000; and 13,000 cells/cm\({}^{2}\)) and ratios as previously described for maximum density. Cells were plated, allowed to rest for 24 h, and then used per manufacturer instructions and as previously described ([PERSON], [PERSON], [PERSON], and [PERSON], 2018). Menadione (50 \(\upmu\)M; MilliporeSigma, St. Louis, MO, USA) was added to positive control wells and allowed to incubate for 2 h. Luminescence was read on a Synergy H1 Microplate reader (BioTek Instruments, Inc., Winooki, VT, USA). Cellular Morphology Microscopy -Confocal Imaging. Cells were treated and stained as previously described [44]. Briefly, cells were grown in chamber slides (Lab-Tek II, Rochester, NY, USA) for 48 h, permeabilized per manufacturer instructions (Image-It FixPerm kit, Molecular Probes, Eugene, OR, USA), washed, and stained (Actin Green 488 Ready Probes reagent, Invitrogen, followed by MitoTracker\({}^{\circledR}\) Red CMXRos and NucBlue\({}^{\circledR}\) Live Cell Stain Ready Probes reagent, Molecular Probes, Eugene, OR, USA). Slides were fixed with 2 drops of ProLong Diamond Anti-fade Mountain (Molecular Probes) and a coverslip placed and allowed to set for 24 h. Images were obtained using Confocal Laser Scanning Microscope (FV-3000, Olympus Corp., Shinjuku, Japan) and rendered with FV31S-SW Windows 10. Fluorescent quantification was performed using Cell Sens Dimension 2 and Windows 10 software. Images were analyzed in full color where minimum, maximum, and average fluorescence were captured and graphed. All images were analyzed at highest magnification images (60\(\times\) under oil immersion) utilizing Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and Graphpad Prism software (Graphpad Software, San Diego, CA, USA). Cellular Morphology-Scanning Electron Microscopy. Cells were grown in 2-well chamber slides (Lab-Tek II, Rochester, NY, USA).