## 2 Materials and Methods  
Individual Cell Populations. Human colon carcinoma Caco-2 (ATCC, Manassas, VA, USA) were used between the passages of 38-45. Mucus-producing HT29-MTX (Sigma, St. Louis, MO, USA), and human [PERSON]'s lymphoma Raji B (ATCC) cell lines were obtained and used at passages numbers 3-10. Dulbecco's Modified Eagle Medium/F-12 (DMEM/F12; 1:1), RPMI-1640, 10% fetal bovine serum, 10,000 U/mL penicillin, 10 mg/mL streptomycin (MP Biomedicals, Santa Ana, CA, USA), and trypsin-EDTA (Invitrogen, Waltham, MA, USA) were used to maintain cultures. Transwell(r) polycarbonate inserts (i.e., 12 wells, pore diameter of 4 \(\upmu\)m polycarbonate) were purchased from Midland Scientific, Inc., Omaha, NE, USA. Caco-2 and HT29-MTX were grown in separate flasks using DMEM-F12 supplemented with 10% fetal bovine serum and a 1% penicillin and streptomycin cocktail at 37 \({}^{\circ}\)C under a 5% CO\({}_{2}\) water saturated atmosphere. Upon confluency (between 80-90%) cells were harvested from flasks using trypsin-EDTA and subsequently reseeded at 5 \(\times\) 10\({}^{5}\) cells per 75 cm\({}^{2}\) flask with culture media replaced every other day. Raji B cells were similarly cultured as above, with RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. In preparation of acclimating Raji B cells into a triple cell culture model utilizing DMEM/F12, over a 4-day period, RPMI-1640 cell culture medium was replaced by 25% of DMEM/F12 per day. This allowed cells to slowly acclimate to supplemented DMEM/F12 cell culture medium. Triple Cell Culture Model Assembly. Triple cell culture model assembly of Raji B, Caco-2, and HT29-MTX cells was seeded at a ratio of 9:9:1 (unless otherwise stated) with the HT29-MTX and Caco-2 cells on the apical chamber of Transwell(r) inserts and the Raji B on the basolateral compartment. The cells were maintained under identical conditions to the monocultures with medium changes every other day. To assemble the layers, first Raji B cells were plated at a known seeding density with 1.5 mL culture medium. Then, the Transwell(r) was inserted, and Caco-2 cells were then seeded at an equivalent density to Raji B cells onto the insert. HT29-MTX cells were seeded last at a density of 1/9 that of the Caco-2 population at the time of Caco-2 seeding to allow for HT29-MTX cells to embed within the Caco-2 layer for proper in vivo correlation [24; 34; 35; 36; 37]. Total volume was increased to 2 mL (1.5 mL on the well; 0.5 mL on the insert) of DMEM/F12; 1:1 culture medium). Caco-2 cells took approximately 5 days to differentiate with Raji B exposure. Cellular Metabolism. Metabolic activity was measured using the MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolzoolium)) assay (Promega, Madison, WI, USA). Raji B, Caco-2, and HT29-MTX cells were seeded separately and in triple cell culture and incubated for 24 h at 37 \({}^{\circ}\)C in 5% CO\({}_{2}\) environment. MTS solution was added to each cell culture per the manufacturer's directions [38; 39]. After a 2-h 37 \({}^{\circ}\)C incubation, the absorbance was measured at 490 nm using a plate reader (Synergy, BioTek, Winooski, VT, USA). Pro-Inflammatory Response Markers. Pro-inflammatory response markers were measured using an enzyme-linked immunosorbent assay (ELISA) for interleukin 6 (IL-6, Invitrogen, Carlsbad, CA, USA). Raji B, Caco-2, and HT29-MTX cells were seeded separately and in triple cell culture and incubated for 24 h at 37 \({}^{\circ}\)C in 5% CO\({}_{2}\) environment. After incubation, cells were washed with phosphate buffer solution (PBS) (Invitrogen) and lysed with RIPA Buffer (Invitrogen). Lysate was collected and centrifuged at 12,000 rpm. Lysate samples and standards were plated per manufacturers direction, and absorbance was read on a plate reader (Synergy H1, BioTek) at 450 nm (620 nm as a reference wavelength) [40; 41]. Raw data was normalized for protein content which was recommended by the manufacturer (high/low protein controls).