4 nitrophenyl n acetyl β d glucosaminide p nag substrate

BMMCs were cultured with rIL-3 and rSCF in the presence or absence of 500 nM TSA for 1 h or overnight. Cells were activated and supernatants and cell lysates were collected 1 h later. β-hexosaminidase (β-hex) activity was assessed, as previously described (28 (link), 59 (link)). Briefly, cells were washed and supernatants and pellets were collected. Pellets were lysed with 0.5% Triton X-100. Both supernatants and pellets were then treated with 4-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG) substrate (Sigma) for 1 h. Plates were read at 405 nm using a spectrophotometer to determine β-hexosaminidase activity. Data are depicted as percent specific release according to the following formula: (Stimulated supernatants/(supernatant ± pellet)^*100−unstimulated supernatants/(supernatant ± pellet)^*100).

BMMCs were cultured in triplicate with IL-3 and SCF in the presence or absence of curcumin for 24 h as described above. To induce activation, 2 μg/ml DNP-IgE was added to the cells. 24h later, they were activated with 200 ng/ml DNP-BSA for 1h. β-hex activity was assessed as previously described [37 (link)]. Briefly, cells were washed and supernatants and pellets were collected. Pellets were lysed with 0.5% Triton X-100. Both supernatants and pellets were then treated with 4-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG) substrate (Sigma) for 1h. Plates were read at 405nm using a spectrophotometer to determine β-hex activity. Data are depicted as percent of cell content demonstrating β-hex activity.