Cell isolation and stimulation
RA FLSs were obtained from the synovial tissues of RA as described
previously (L. Wang et al., 2016) and cells were cultured in a medium of
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and 1% antibiotics. RA FLSs between passages 4 and 7
were maintained for further experiments. Human peripheral blood
mononuclear cell (PBMC) were isolated from venous blood of RA patients
by density gradient centrifuging with Lympholite/H. The gradients were
spun at 800 g ×10 minutes at 22 °C. PBMC cells at the interface were
removed, washed twice and resuspended in serum-free medium (RPMI 1640).
The PBMC preparations were then ready for AZM treatment. AZM and
Etanercept (ETC), dissolved in dimethyl sulphoxide at a concentration of
1 mM, was brought to the final concentration with complete medium.
Before treatment with AZM and ETC, RA FLSs and PBMC were cultured
overnight in medium containing 1% fetal cells and subsequently
stimulated with IL-1β (#8900, Cell Signaling Technology, USA) and TNF-α
(#7321, Cell Signaling Technology, USA), or lipopolysaccharides (LPS,
Sigma, St. Louis, MO, USA).