Cell cultures
Splenocytes were placed in 48-well tissue culture plates at a density of
5×105 cells/well with or without SBI (0, 20, 50, 100,
200μM) (dissolved in DMSO), and the control was a DMSO solution without
SBI. After incubation for 24h at 37℃ in 5% CO2, the
supernatant and the cells were collected for immediate assay or stored
at -80℃. To verify the effect of MSCs on mice splenocytes, they were
placed in 24-well tissue culture plates at a density of
6×104 cells/well with or without SBI
(100μM)
and incubated at 37 °C with 5% CO2 for 4 hours, and
then 5×105 splenocytes were added. 24 hours later, the
supernatant and cells were collected for immediate assay or stored
at
-80℃.
To confirm the impact of interleukin (IL) -6, STAT3 and mammalian target
of rapamycin (mTOR) pathways on SBI efficacy, STAT3 agonist colivelin
(MCE, USA) (50nM) and inhibitor
Stattic (MCE, USA) (20μM), mTOR
agonist MHY1485 (MCE, USA) (100nM)
and inhibitor Rapamycin (MCE, USA) (100nM) as well as IL-6 (Genscript
BioTECH, USA) (20 ng/ml) were added into mouse splenocyte cultures
respectively with or without SBI and incubated for 24 hours. The above
stimulant concentrations were provided as a reference by MCE
Biotechnology and were used after in vitro validation.