Versamax microplate spectrophotometer

The degranulation was measured by β-hexosaminidase release as described previously [51 (link)]. Briefly, transfected RBL-2H3 cells (5 × 10⁴ cells per well) were seeded into a 96-well, white, clear-bottom cell culture plate and incubated overnight in a 37 °C incubator with 5% CO₂. To determine the inhibitory effects of PTx and YM-254890 on MC degranulation, cells were pretreated with PTx (100 ng/mL, 16 h) and/or YM-254890 (10 μM, 5 min) prior to stimulation with SP. Cells were then washed twice and suspended in a total volume of 50 μL HEPES buffer containing 0.1% bovine serum albumin (BSA). Experimental groups were stimulated with SP for 30 min at 37 °C. Cells without treatment were designated as controls. To determine the total β-hexosaminidase release, unstimulated cells were lysed in 50 μL of 0.1% Triton X-100. Aliquots (20 μL) of supernatants or cell lysates were incubated with 20 μL of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) for 1 h at 37 °C. The reaction was stopped by adding 250 μL of stop buffer (0.1 M Na₂CO₃/0.1 M NaHCO₃). The β-hexosaminidase release was assessed by measuring absorbance at 405 nm using Versamax microplate spectrophotometer (Molecular Devices, San Jose, CA, USA).

The quantification of starch was carried out by digestion with amylolytic enzymes (amyloglucosidase and amylase) and subsequent quantification of reducing sugars [54 (link)]. Previously, free sugars were removed with successive 80% ethanol washings. After digestion, an aliquot was assayed with 3,5-dinitrosalicylic acid (DNS) and Na/K tartrate measuring the absorbance at 570 nm in a VERSA max microplate spectrophotometer (Molecular Devices, San Jose, CA, USA), using glucose for the standard curve.

RBL-MRGPRX2 cells (5×10⁴ cells/well), LAD2 cells (1×10⁴ cells/well), primary human skin-derived MCs (5×10³ cells/well), and PMCs (1×10⁴ cells/well) were seeded into a 96-well plate in a total volume of 50 μl HEPES buffer containing 0.1% bovine serum albumin (BSA). Cells were preincubated in the absence or presence of MRGPRX2 antagonist for 5 min followed by the addition of SP, PAMP-12, or rocuronium for 30 min at 37°C. To determine the total β-hexosaminidase, cells were lysed in 50 μl of 0.1% Triton X-100. Aliquots (20 μl) of supernatants were incubated with 20 μl of 1 mM p-nitrophenyl-N-acetyl-b-D-glucosamine (PNAG) at 37°C for 1 h (RBL-MRGPRX2 cells), and 1.5 h (LAD2, primary human skin-derived MCs, and PMCs). Finally, 250 μl of stop solution was added (0.1 M Na₂CO₃/0.1 M NaHCO₃) to stop the reaction. The absorbance was measured with a microplate reader at a wavelength of 405 nm using a Versamax microplate spectrophotometer (Molecular Devices, San Jose, CA). Percentage of β-hexosaminidase degranulation was calculated by dividing the β-hexosaminidase release in the sample by total β-hexosaminidase release (37 (link)).

DPPH radical scavenging activity was evaluated using the method of Blois [36 (link)], with slight modification. Test samples and DPPH were dissolved in methanol. Each test sample (160 µL) at various concentrations was added to 40 µL of DPPH solution (1.5 × 10⁻⁴ M). After gently mixing and standing at room temperature for 30 min, the optical density of the reactant was measured at 520 nm using a VERSAmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The antioxidant activity of samples is expressed in terms of EC₅₀ values (μM required to scavenge DPPH radical by 50%), which was calculated from the log-dose inhibition curve. L-Ascorbic acid was used as a positive control.