Gemini em microplate spectrofluorometer

Evans Blue dye, 961 Da, was used as a tracer for assessing BBB disruption. The Evans Blue extravasation assay was performed as previously described (Borlongan et al., 2004b (link); Garbuzova-Davis et al., 2013 (link)). Briefly, after perfusion, rat brains were divided into right and left hemispheres. Brain tissues were weighed and placed in 50% trichloroacetic acid solution (Sigma). Following homogenization and centrifugation, the supernatant was diluted with ethanol (1:3) and loaded into a 96 well-plate in triplicates. Sera were diluted with ethanol (1:10,000) and loaded separately into a 96 well-plate in triplicates also. The dye was measured with a spectrofluorometer (Gemini EM Microplate Spectrofluorometer, Molecular Devices) at excitation of 620nm and emission of 680nm (Ay et al., 2008 (link)). Calculations were based on external standards in the same solvent. The tissue Evans Blue content was quantified from a linear standard curve derived from known amounts of the dye and was normalized to tissue weight (µg/g). For sera, Evans Blue concentration was quantified similarly and presented as µg/ml. All measurements were performed by two experimenters blinded to the experiment.

5 × 10⁴ hMCs or 1 × 10⁵ mMCs were pretreated with BoNT A or B (0.5-5 pM) for 24 hours. After being washed 2 times with PBS, MC degranulation was assessed by measuring the activity of β-hexosaminidase in the supernatants[39 (link)-41 ] of 1 × 10⁵ mMCs 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) or 5 × 10⁴ hMCs in 100 μl saline phosphate buffer (0.9 % NaCl, 10 mM NaH₂PO₄, 45 mM glucose) incubated for 30 min at 37°C with Compound 48/80 (10 μg/ml, Sigma) which was used to promote MC degranulation in an IgE-independent manner[42 (link)]. For each sample assayed, MC supernatant aliquots (20 μl) were mixed with substrate solution (100 μl), which consisted of 1 mM 4-methylumbelliferyl-2-acetamide-2-deoxy-β-D-glucopyranoside (Calbiochem) in 0.1 M sodium citrate buffer (pH 4.5), and were incubated for 2 hours at 37°C in the dark. The reaction was then stopped by the addition of 12 μl of 0.2 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). To determine the total cellular content of this enzyme, an equivalent number of cells were lysed with 1% triton-X-100 (Sigma). Release of β-hexosaminidase was calculated as the percentage of the total enzyme content.

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.