Penicillin streptomycin solution ps

Human PBMCs were isolated from venous blood using Histopaque-1077 (Sigma-Aldrich, Saint Louis, MO, USA), as described.61 (link) For differentiation to macrophages, 1 mL aliquots containing 2×10⁷ PBMCs in RPMI supplemented with 1% (v/v) penicillin-streptomycin solution (PS, Sigma-Aldrich) were seeded in flat bottom 12-well plates and incubated 1.5 h at 37°C, 5% (v/v) CO₂. Non-adherent cells were discarded, and the remaining adherent cells were washed with PBS at 37°C, and 1 mL of X–VIVO serum-free medium (Lonza, Basel, Switzerland) supplemented with 1% (v/v) PS and 10 ng mL⁻¹ recombinant human granulocyte-macrophage colony-stimulating factor (Sigma-Aldrich) were included per well.62 (link) For differentiation to immature dendritic cells (DCs), adherent cells were added 1 mL X–VIVO serum-free medium supplemented with 1% (v/v) PS, 500 U mL⁻¹ recombinant human IL-4 (Sigma-Aldrich), and 800 U mL⁻¹ recombinant human granulocyte-macrophage colony-stimulating factor (Sigma-Aldrich) for 6–7 days.63 (link),64 (link) In both cases, plates were incubated for 6–7 days at 37 °C, 5% (v/v) CO₂, replacing the medium every 2 days. Differentiation to DC cells was confirmed by flow cytometry, detecting DC-SIGN with FITC-conjugated mAb anti-human DC-SIGN (Thermo-Fisher Scientific, Waltham, MA, USA), as described elsewhere.63 (link)

Human intestinal epithelial Caco-2 cells and mouse embryonic fibroblast (MEF) cell lines (American Type Culture Collection, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% Penicillin-Streptomycin solution (P/S, Sigma-Aldrich Co., USA) at 37℃ under humidified 5% CO₂. Caco-2 polarization was performed as follows: 24-well Transwell (35024, SPL, Korea) dishes were coated with 396 µg/cm² collagen, seeded with Caco-2 cells, and monolayers were grown for 10 days. The experiment was performed when the Transepithelial/Transendothelial Electrical Resistance (TEER) reached a value in the range of 500–600 Ωcm². LF stock solution (100 mg/mL) was made by dissolving 100 mg of the dry crude extract in 1 mL DMSO. Cell viability was determined by the WST assay kit (EZ-cytox, Daeil lap service Co. Ltd., Seoul, Korea). Caco-2 cells were seeded in 96-well plate and incubated for 24 hours (h) under the same culture conditions previously mentioned. Cells were pretreated with various concentrations of LF (0, 12.5, 25, 50, 100 and 250 µg/mL) for 24 h followed by WST assay, according to the manufacturer's instruction. Absorbance was measured at 450 nm on a micro-plate reader (xMark™ Microplate Absorbance Spectrophotometer, Bio-Rad Inc., Hercules, California, USA) after 3 h.