Transwell insert plate

Caco-2 cells were subjected to cultivation in MEM medium encompassing 1% glutamax, 1%, non-essential amino acids, 1% sodium pyruvate 100 mM Solution (all from Gibco, U.S.A.), and 20% FBS (Hyclone, U.S.A.). These cells were cultivated under the conditions of 5% CO₂ and 95% air at 37 °C.
Caco-2 cells were subjected to 21-day cultivation in transwell insert plates (1.5 × 10⁵ cells/cm²; available from Corning, Cambridge, MA, USA), and a targeted cell monolayer with a transepithelial electrical resistance value (TEER) was acquired with a Millicell ERS-2 V-ohm meter (available from Millipore Corporation, Billerica, MA, USA) [20] (link).
Caco-2 cells were subjected to 12-h treatment with LPS (10 μg/mL, Sigma-Aldrich) to establish intestinal barrier damage model. Subsequently, Caco-2 cells were treated for 24 h with digestive products of CSP (CSP-DP) (20 μM) in intestinal barrier damage model to validate the nutritional quality.

A co-culture system was established as described previously [21 (link)]. Briefly, Caco-2 cells (passage numbers: 48 - 62) were seeded onto Transwell insert plates (3.75 × 10⁵ cells/well; Corning Costar Corp., Cambridge, MA, USA) and incubated for 21 d to obtain an integrated cell monolayer with a transepithelial electrical resistance value (TER) 1,200 Ω cm² using a Millicell-ERS instrument (Millipore Corporation, Billerica, MA, USA). Cell culture medium was changed every 3 d. RAW 264.7 macrophages (passage numbers: 10 - 30) were seeded in 6-well tissue culture plates (8.5 × 10⁵ cells/well) and incubated overnight. After replacing the media with RPMI1640, transwell inserts containing Caco-2 cells were added to the 6-well plates containing RAW 264.7 macrophages (Fig. 1). To evaluate the anti-inflammatory effects of SQE in this co-culture system, various concentrations of SQE were applied to the apical side of the transwell insert for 3 h, followed by the addition of 1 µg/ml lipopolysaccharide (LPS, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to the basolateral side. After an additional incubation overnight, culture supernatants from the basolateral side were collected to measure levels of nitric oxide (NO), PGE₂, IL-6, and IL-1β. The cultured cells were subsequently harvested to isolate total RNA for RT-PCR and western blot analyses.

Briefly, the Caco-2 cells were seeded at 3.0 × 10⁵ cells/well onto Transwell insert plates (1.12 cm², 0.4 µm pore size [Corning Costar]). The cells were fully differentiated (TER value > 400 Ω·cm²), and were subjected to the following experiment. RAW264.7 cells were seeded at 2.1 × 10⁵ cells/well in 24-well tissue culture plates and incubated overnight to fully adhere to the well. After all media had been replaced with RPMI 1640 (Gibco BRL), the Transwell insert plates with the Caco-2 cells were added to the wells of multiple plates preloaded with RAW264.7 cells. Then, 100 µg/ml of EPSs purified by several strains and a whey sample were applied to the apical side and incubated at 37°C for 3 hr. After 3 hr of the incubation, LPS [Wako] was added to the basolateral side. After 3 hr of the incubation, all culture supernatants from the basolateral side were collected for TNF-α measurement. The treated Caco-2 cells and
RAW264.7 cells were then harvested to isolate total RNA for real-time polymerase chain reaction assays as described below.

Roswell Park Memorial Institute 1640 (RPMI 1640) and fetal bovine serum (FBS) were purchased from Vitrocell (Sao Paulo, Brazil). Ketamine and xylazine were obtained from Ceva Santé Animale (Paulínea, Brazil). Oyster glycogen, propidium iodide (PI), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), trypan blue, CoCl₂ and Ringer–Locke solution were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Anti-Ly6G antibody conjugated with phycoerythrin (PE) and annexin V (AnxV)–fluorescein isothiocyanate (FITC) were purchased from BD Biosciences (San Jose, CA, USA). Percoll was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Enzyme-linked immunosorbent assay (ELISA) kits were obtained as follows: TGF-β and IL-10 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA); VEGF-A was obtained from IBL (Mikasa-Shi, Japan) and ELISA, arginase-1 and MMP-9 were obtained from MyBioSource (San Diego, CA, USA). Transwell insert plate was purchased from Costar (Cambridge, MA, USA).