Premix wst 1 cell proliferation assay system

Lymphatic endothelial cells and human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. Lymphatic endothelial cells were cultured in endothelial cell growth medium‐2MV (No. cc4176; Lonza). HUVECs were cultured in endothelial cell growth medium plus growth media (No. cc5035; Lonza). Cells (10 000/well) were plated in 96‐well plates and maintained in corresponding media until 80% confluent. Cells were then maintained in endothelial cell basal medium‐2 containing 0.5% fetal bovine serum for 24 hours. Cells were then exposed to 100 ng/mL of VEGF‐C_Cys156Ser or VEGF‐A. Cell proliferation was evaluated 72 hours later by use of the PreMix WST‐1 Cell Proliferation Assay System (TAKARA Bio Inc), according to the manufacturer's instructions.

The proliferation of HLE and HuH7 cells was evaluated using a Premix WST-1 Cell Proliferation assay system (Takara Bio Inc., Kusatsu, Japan). After transfection of siPRMT5, HLE and HuH7 (3×10³ cells/well) cells were seeded into 96-well plates in DMEM containing 2% FBS. The optical density of each well was measured 1 h after the addition of 10 μl of WST-1 0, 1 and 3 days after seeding (23 (link)). The growth rate indicated an incremental rate of the optical density from the cell seeding day.

In total, 10⁴ cells were infected with rMV‐SLAMblind at an MOI of 2 and then cultured in 96‐well plates. The cell viability was determined using a Premix WST‐1 Cell Proliferation Assay System (TaKaRa) at the indicated time points. The cell viability was calculated by dividing the average value of nonviral infected cells by 100.

Human LX2 HSCs (3×10³ cells/200 µl) were seeded into a 96-well plate and cultured in Dulbecco's Modified Eagle's Medium (Nacalai Tesque, Inc.) supplemented with 10% fetal bovine serum (Cat. No. FB-1365/500; Biosera, Kansas, USA) and 0.01% penicillin-streptomycin-L-glutamine solution in a 5% CO₂ humidified atmosphere at 37°C. The growth medium was refreshed every other day. Cell viability was investigated using a WST-1 assay (cat. No. MK400; Premix WST-1 Cell Proliferation Assay System; Takara Bio, Inc., Otsu, Japan) in the presence of periostin and/or losartan, according to the manufacturer's protocol. Briefly, cells were incubated for 24 h before the addition of media containing periostin (0.1 or 1.0 µg/ml; Cat. No. C-60045; PromoCell GmbH, Heidelberg, Germany) or Losartan (10 µM; Cat. No. 12353-04; Nacalai Tesque, Inc.). Cells were incubated for a further 48 h and then analyzed using a WST-1 assay. WST positivity was assessed using a plate reader at 450 nm.

Docetaxel and Nanoxel were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Samyang Biopharmaceuticals (Seongnam, Korea), respectively. Thrombin, 5-hydroxytryptamine (5-HT), menadione, and butylated hydroxyanisole (BHA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Collagen was acquired from Chrono-log (Havertown, PA, USA) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) was obtained from Seoul Clinical Genomics (Seoul, Korea). Fura-2/AM and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) were purchased from Molecular Probes (Eugene, OR, USA). Premix WST-1 Cell Proliferation Assay System was acquired from Takara Bio (Shiga, Japan). All other chemicals used were of the highest purity available and purchased from standard suppliers.