Human nucleus pulposus cells

Human nucleus pulposus cells were purchased from ScienCell. Cells were incubated in human NP cell medium (ScienCell) with fetal bovine serum and antibiotics. When the confluence reached about 70%-90%, the cells were trypsinized, counted, and passaged. Cells from passages 4 to 7 were cultured in 6-well plates with about 8 ml medium. When they adhered, the medium was changed and the following treatments were performed.

The human nucleus pulposus cells used in this experiment were purchased from ScienCell. After defrosting, cells were inoculated into tissue culture dishes and cultured in DMEM complete medium supplemented with 1% penicillin-streptavidin, 1% L-glutamine, and 10% fetal calf serum in a 37°C humidified incubator. The cells were subcultured at a dilution of 1 : 3 when they reached 90% confluency. To test the induction of COX2 by IL-1β, cells at P3 were subjected to the addition of IL1β at the final concentration of 0, 5, 10, and 15 ng/ml for 24 hr. After 24 hr, the cells were harvested. RNA was isolated by using Trizol and assessed by using a Nanodrop bioanalyzer (Thermo Scientific, US). Reverse transcription of RNA to cDNA was done with an RNA to cDNA kit (Tsingke, PRC). Quantitative real-time PCR (qRT-PCR) of the expression of COX2 was performed on a StepOnePlus system (Applied Biosystems, Life technologies, US) using SYBR green real-time PCR master mixes (Tsingke, PRC). GAPDH was tested as an endogenous control. The relative quantification was achieved by the comparative CT method.