Infinite m200 fluorescence microplate reader

OVA‐specific serum IgG1, IgG2a, IgE and IgA levels were determined by ELISA. The 96‐well microtitre plates were coated with OVA (5 μg ml⁻¹). Serum samples were diluted 1:10,000 for IgG1, 1:100 for IgG2a, 1:10 for IgE and 1:10 for IgA. Rat anti‐mouse IgG1, IgG2a, IgE and IgA antibodies (2 μl ml⁻¹; Pharmingen, San Diego, CA USA) were applied, followed by peroxidase‐conjugated mouse anti‐rat IgG antibodies (1:2000; Jackson Immuno Labs, West Grove, PA, USA). Antibody levels were quantified based on optical densities.
The allergen‐specific IgE levels in sera were quantified by analysing the degranulation of rat basophil leukaemia (RBL‐2H3) cells (Wiedermann et al., 2001). RBL‐2H3 cells were plated in 96‐well tissue culture plates (4 × 10⁴ cells per well) and sensitized by incubation with mouse serum (diluted 1:270 or 1:810) for 2 h. The cells were washed, OVA (0.6 mg ml⁻¹) was added, and the cells were incubated for 30 min at 37°C for degranulation. Supernatants were recovered and incubated with 4‐methylumbelliferyl‐N‐acetyl‐β‐D‐glucosaminide (Sigma‐Aldrich, St Louis, MO, USA), and β‐hexosaminidase was analysed using an Infinite M200 fluorescence microplate reader (λex:360 nm/λem:465 nm; Tecan Group, Grodig, Austria). The results are reported as the percentage of total β‐hexosaminidase released from cells after disruption with 1% Triton X‐100.

Cell viability was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. In brief, cells were incubated with 20 μL of MTT (0.5 mg/mL) in 96-well plates, and the supernatant was discarded. Dimethyl sulfoxide (150 μL) was added to the wells, which were then rotated for 10 min to dissolve formazan. Absorbance was measured at 540 nm using an Infinite M200 fluorescence microplate reader (Tecan Group AG., Männedorf, Switzerland).

BODIPY 493 (Thermo Fisher Sci., Waltham, MA) was used to stain lipid droplets. For confocal microscopy imaging, BODIPY 493 was diluted with PBS to a final concentration of 1.25 μg/mL from its stock (1 mg/mL in 100% ethanol). Desiccated cells were briefly rehydrated and stained directly with the diluted BODIPY 493 for 5 min in the dark. Stained cells were imaged using a Zeiss 700 laser scanning confocal microscope (Zeiss, NY) with a 63X, 1.4 NA oil objective. Z-stacks of 1 μm step size were acquired and images were presented as maximum projections for fluorescence and the middle section for bright-field. For counting the number of LDs, at least 70 cells were counted from the maximum projected images of each experiment. Data were presented as mean ± standard derivation (SD) from 3 independent experiments.
For quantitative comparison of the LD fluorescence intensity, cells were first diluted and adjusted to OD₆₀₀ = 0.1. Cells were then stained with BODIPY 493 for 5 min in the dark. The relative fluorescence intensity was measured using a Tecan infinite M200 fluorescence microplate reader (Tecan US, Morrisville, NC) with excitation/emission at 495/530 nm. Data were presented as mean ± standard derivation (SD) from 3 independent experiments.