Degranulation percentage was assessed by measuring the activity of β-hexosaminidase in the supernatants of 1×105 MCs in 200 ml Tyrode's buffer incubated for 1 hr with 0 or 20 nM of Cath LL-37, 1% Triton X-100 and 10 μg/mL compound 48/80 (Sigma-Aldrich, St Louis, MO) as a positive control. For each sample assayed, supernatant aliquots (25 μL) were mixed with substrate solution (50 μL), which consisted of 10 mM 4-methylumbelliferyl-2-acetamide-2-deoxy-b-D-glucopyranoside (Calbiochem; EMD Millipore, Billerica, MA) in 0.1 M sodium citrate buffer (pH 4.5), and were incubated for 30 min at 37°C. The reaction was then stopped by adding 50 μL of 0.4 M glycine (pH 10.7). The reaction mixtures were excited at 365 nm and measured at 460 nm in a fluorescence plate reader (Gemini EM microplate spectrofluorometer; Molecular Devices, Sunnyvale, CA). To determine the total cellular content of this enzyme, an equivalent number of cells were lysed with 1% Triton X-100 and 10 μg/mL compound 48/80 (Sigma-Aldrich). Release of β-hexosaminidase was calculated as the percentage of the total enzyme content.
4 methylumbelliferyl 2 acetamide 2 deoxy b d glucopyranoside
Manufactured by Merck Group
Sourced in United States
4-methylumbelliferyl-2-acetamide-2-deoxy-b-D-glucopyranoside is a chemical compound used as a fluorogenic substrate for the detection and measurement of enzymatic activity, particularly related to glycosidases. It is a synthetic derivative of 4-methylumbelliferone, which acts as a fluorescent reporter upon enzymatic cleavage.
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3 protocols using 4 methylumbelliferyl 2 acetamide 2 deoxy b d glucopyranoside
1
Assessing Mast Cell Degranulation
2
Mast Cell Degranulation Assay
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Degranulation was assessed by measuring the activity of β-hexosaminidase in the supernatants of 1 × 105 MCs in 200 µL Tyrode’s buffer (0.1% BSA, 0.1% glucose, 2 mmol/L MgCl2, 137.5 mmol/L NaCl, 12 mmol/L NaHCO3, 2.6 mmol/L KCl, pH 7.4) incubated for 24 h with 0.5 U of BoNT-A1 or -B1 before the addition of 1 μg/mL 48/80 (Sigma Aldrich, St Louis, MO, USA). For comparison of mast cell degranulation by 48/80 to CQ, 10 μg/mL 48/80 was used. For each sample assayed, supernatant aliquots (20 μL) were mixed with substrate solution (100 μL) which consisted of 10 mM 4-methylumbelliferyl-2-acetamide-2-deoxy-b-d -glucopyranoside (EMD Millipore, Billerica, MA, USA) in 0.1 M sodium citrate buffer (pH 4.5) and were incubated for 2 h at 37 °C in the dark. The reaction mixtures were excited at 365 nm and measured at 460 nm in a fluorescence plate reader (Gemini EM microplate spectrofluorometer; Molecular Devices, Sunnyvale, CA, USA). To determine the total cellular content of this enzyme, an equivalent number of cells were lysed with 1% Triton X-100 (Sigma Aldrich, St Louis, MO, USA). Release of β-hexosaminidase was calculated as the percentage of the total enzyme content.
3
Assessing Mast Cell Degranulation
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