Mast cell activation was assessed by β-hexosaminidase release in BMMCs84 (link),85 (link). In short, BMMCs were inoculated into 96 well plates with 8*103 cells per well and sensitized with 0.1ug/ml IgE (D8406, Sigma-Aldrich) prepared with serum-free medium. After sensitization for 24 h, the culture medium was discarded and LPS (L2630, Sigma-Aldrich) complete culture medium was added. After 6 h, the medium was discarded and 1 ug/ml DNP-HSA (D-5059, Biosearch Technologies) was added for 30 min.
The supernatant of each well was collected, and NP40 Lysis Buffer was added to each well to lyse the cells for 5 min. Lysate (50 ul) and supernatant (50 ul) were each incubated with substrate solution (50 μL) (3 mM 4-Nitrophenyl N-acetyl-β-D-glucosaminide, N9376, Sigma-Aldrich) for 90 min at 37 °C, then 150 uL of Na2CO3/NaHCO3 (pH = 10.6) were added to terminate the reaction, and the absorbance value was read at 405 nm by Cytation 5 multifunctional enzyme marker (BioTek, USA), and the degranulation ratio of mast cells was obtained by supernatant/(supernatant + lysate) absorbance value.
The supernatant of each well was collected, and NP40 Lysis Buffer was added to each well to lyse the cells for 5 min. Lysate (50 ul) and supernatant (50 ul) were each incubated with substrate solution (50 μL) (3 mM 4-Nitrophenyl N-acetyl-β-D-glucosaminide, N9376, Sigma-Aldrich) for 90 min at 37 °C, then 150 uL of Na2CO3/NaHCO3 (pH = 10.6) were added to terminate the reaction, and the absorbance value was read at 405 nm by Cytation 5 multifunctional enzyme marker (BioTek, USA), and the degranulation ratio of mast cells was obtained by supernatant/(supernatant + lysate) absorbance value.
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