Dy410

TNF-α release was measured in cell supernatants by ELISA (DY410, R&D Systems) according to the manufacturer’s instructions.

Macrophage colony‐stimulating factor differentiated bone marrow‐derived macrophages were plated in 1500 μL/well in 10% FCS, 1% PS in RPMI. Medium was either supplemented with 10 or 50 ng/mL lipopolysaccharide (Sigma, K235). Cells were cultured for 24 hours and thereafter, medium was collected for ELISA as well as aortic ring assay, after which Trizol was added for RNA isolation. Supernatant was stored at −80 °C. CCL2 (C‐C motif chemokine ligand 2), IL‐6 (interleukin‐6), TNF‐α (tumor necrosis factor alpha), and VEGF concentrations were determined by ELISA according to manufacturer's protocol (R&D systems, DY406, DY410, DY479, and DY493).

To compare and verify probiotic BEV anti-inflammatory bioactivity between isolation methods, we utilized a mouse macrophage stimulation assay ^{19 (link)}. RAW264.7 mouse macrophages were seeded at 75,000 cells/well in a 48-well plate in DMEM + 5% FBS + 1% penicillin/streptomycin (Media). Then, 24 h later, cells were pretreated with media supplemented with either: i) 1× PBS (6 wells total), ii) BEV-depleted conditioned media (permeate from TFF), iii) 1 μg/mL dexamethasone as a positive control (Dex; D4902-25 MG; Sigma-Aldrich), and iv) BEV groups from various isolation methods (TFF only, TFF + SEC, or TFF + HPAEC). All groups were performed in triplicate and doses between BEV-depleted conditioned media and BEV groups were normalized by protein content. After 24 h of incubation, the pre-treatments were removed and 10 ng/mL LPS (resuspended in 1× PBS; L4391-1MG; Sigma-Aldrich) was added to all groups except three PBS pretreated wells (media only group) to stimulate inflammatory responses. Conditioned media was collected 4 hours post-LPS stimulation, stored at −80°C, and levels of tumor necrosis factor-α (TNF-α) were assessed by ELISA within 3 days (DY410; R&D Systems). The cytokines TNF-α, IL-6, and IL-10 were previously screened and TNF-α was selected due to a high dynamic range of dose-dependent suppression following BEV pretreatment.