Increase in H₂S concentration in culture medium after differentiation.

Confluent 3T3L1 cells were washed with PBS (37°C) and resuspended in normal growth DMEM for 10 min. Aliquots of medium were collected (‘baseline’ group). 3T3L1 cells were then cultured for 2 days either in DMEM (‘negative control’ group) or differentiation medium (‘positive’ control group). To test the effect of H₂S releasing drugs, 3T3L1 cells were co-incubated with GYY4137 (50 μM) or NaHS (50 μM) for 2 days. H₂S concentration in the medium was detected using a 2,6-dansyl azide fluorescent probe. Statistical significance was determined by ANOVA followed by Fisher's LSD posthoc analysis. Data shown are mean±SEM of 4 independent experiments. *P < 0.05 vs. baseline group; &P < 0.05 vs. 48 h negative control group.