Endothelial cell growth medium 2 kit

Pooled primary Human umbilical vein endothelial cells (HUVECs) were purchased from Promocell (C-12203). Cells were cultured in endothelial cell basal medium supplemented with endothelial cell growth medium 2 kit (Promocell; C-22111) at 5% CO2 and a temperature of 37°C. All cells were grown on 0.1% (w/v) gelatin-coated cultureware and were not used in excess of four passages.

HUVECs were cultured using the Endothelial Cell Growth Medium 2 Kit (C-22111, PromoCell, Heidelberg, Germany). The cells were incubated with or without TNFα (10 ng/mL), Irisin (10 nM), and BAIBA (40 µM) for 24 hours. After incubation, proteins extracted from cultured HUVECs were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for one hour at room temperature, incubated with primary antibodies including anti-VCAM-1 (1:1000, #ab134047, abcam) and anti-MCP-1 (1:1000, #ab9669, abcam) antibodies overnight at 4 °C, washed with TBS-T three times, and incubated with secondary antibody (1:2000, #7074, Cell Signaling Technology Japan K.K., Tokyo, Japan) for one hour at room temperature. The membranes were washed with TBS-T three times and signals were then detected using a chemiluminescence kit (RPN2236, GE Healthcare Japan, Tokyo, Japan), C-DiGit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA), and Image Studio Software (LO-COR Biosciences).

HeLa, A549, and THP-1 cell lines were purchased from ATCC, the HaCat cell line was purchased from Cell Lines Service, HUVECs and NHEKs were purchased from PromoCell, and Caco-2 cells were purchased from the Riken Cell Bank. HeLa and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Gibco) and 50 μg/ml gentamicin (Nacalai Tesque), and the THP-1 cells were cultured in RPMI 1640 medium (Nacalai Tesque) supplemented with 10% FBS and 50 μg/ml gentamicin. THP-1 cells were differentiated into macrophages by stimulating them with 50 ng/ml phorbol 12-myristate for 72 h. HUVECs were maintained with the endothelial cell growth medium 2 kit (PromoCell) supplemented with 10% FBS and 50 μg/ml gentamicin. NHEKs were cultured with the keratinocyte growth medium 2 kit (PromoCell), and Caco-2 cells were maintained in minimum essential medium (Wako) supplemented with 10% FBS and 50 μg/ml gentamicin. Cells were incubated in a 5% CO₂ incubator at 37°C.