Sparkcontrol magellan plate reader

RBL cell mediator release assay was performed as described elsewhere [19 (link)]. Briefly, RBL 2H-3 (ATCC, Manassas, VA, USA) cells were plated into 96-well plates (4 × 10⁴ cells/well) and incubated with serum samples (1:300) from days −11 and 25 for 2 h at 37 °C in a 5% CO₂ atmosphere. Cells were washed with Tyrode’s buffer (137 mM NaCl, 5.6 mM d-glucose, 2.7 mM KCl, 1.8 mM CaCl₂, 1.1 mM MgCl₂, 0.4 mM NaH₂PO₄, 12 mM NaHCO₃, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% (w/v) BSA; pH = 7.4; Sigma-Aldrich) and degranulation of cells was induced by incubation with 0.3 µg/mL OVA in Tyrode’s buffer. Supernatants were analyzed for β-hexosaminidase content by incubation with 80 μM 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (Sigma-Aldrich) and measuring fluorescence at λ_ex: 360 nm/λ_em: 465 nm with a SparkControl Magellan plate reader. Results show the percentage of total β-hexosaminidase release in serum samples compared to positive controls after adding 1% (v/v) Triton X-100 (Sigma-Aldrich) in double-distilled water.

RBL cell mediator release assay was performed as described elsewhere (41 (link)). Briefly, RBL 2H-3 cells were plated into 96-well plates (4 x 10^4 cells/well) and incubated with serum samples (1:300) from the beginning and the end of the experiment for 2 h at 37°C in a 5 % CO₂ atmosphere. Cells were washed with Tyrode’s buffer [137 mM NaCl, 5.6 mM D glucose, 2.7 mM KCl, 1.8 mM CaCl₂, 1.1 mM MgCl₂, 0.4 mM NaH₂PO₄, 12 mM NaHCO₃, 10 mM HEPES and 0.1 % (w/v) BSA; pH = 7.4; Sigma-Aldrich] and degranulation of cells was induced by incubation with 0.3 µg/ml OVA in Tyrode’s buffer. Supernatants were analyzed for β-hexosaminidase content by incubation with 80 μM 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (Sigma-Aldrich) and measuring fluorescence at λex: 360 nm/λem: 465 nm with a SparkControl Magellan plate reader. Results show percentage of total β-hexosaminidase release after adding 1 % (v/v) Triton X-100 (Sigma-Aldrich) in ddH₂O.