*2.7. MTT Assay*

RBL-2H3 cells (5 × 10<sup>4</sup> cells per well) cells were plated in 96-well plates and then grown in medium containing 10% FCS at 37 ◦C in a humidified 5% CO2 incubator. After 16 h, cells were washed with Tyrode's Buffer (137 mM NaCl, 2.7 mM KCl, 1mM MgCl2, 1.8 mM CaCl2, 0.2 mM Na2HPO4, 5.5 mM Glucose, 10 mM HEPES, and 0.1% BSA), and then incubated with LiRecTCTP (10, 50, and 100 μg/mL), total venom from *L. intermedia* (10, 50, and 100 μg/mL), 48/80 compound (100 μg/mL) (positive control for degranulation), and PBS or the recombinant toxin LiRecDT1H12A (100 μg/mL) (as negative controls). After 2 h, media was removed and replaced by MTT solution (0.5 mg/mL) (Sigma Aldrich). It is important to mention that incubation with toxins did not cause any detachment of cells from the plates. Cells were again incubated for 3 h at 37 ◦C. The MTT solution was removed, and formed formazan crystals of each sample were solubilized with DMSO (100 μL) (Sigma Aldrich). The dehydrogenases activity for cell viability assessment was quantified spectrometrically in 550 nm. MTT assay was performed in pentaplicate, and the results are shown as mean ± s.d. of three independent experiments.

### *2.8. In vitro Mast Cell Degranulation Induced by LiRecTCTP*

The release of granular beta-hexosaminidase enzyme was measured in the supernatants obtained from RBL-2H3 rat cell line exposed to the recombinant toxins. For this, 5 × 10<sup>4</sup> cells were plated in medium with 10% FCS. After 16 h, cells were washed, and the medium was replaced by Tyrode's buffer containing LiRecTCTP (10, 50, and 100 μg/mL) with or without cromolyn (10 μM), total venom of *L. intermedia* (10, 50, and 100 μg/mL), 48/80 compound (100 μg/mL) (positive control), and PBS or the recombinant toxin LiRecDT1H12A (100 μg/mL) (as negative controls) for 2 h at 37 ◦C in a humidified 5% CO2 incubator. From each experimental sample to be quantified, five aliquots (10 μL) of the supernatants were taken as pentaplicates to another microwell plate. RBL-2H3 cells incubated only with the Tyrode's (TGB) buffer were lysed with 0.5% Triton X-100 (200 μL) (Sigma Aldrich) to evaluate the total enzyme content as 100% reference. To all replicates, 90 μL of the substrate solution containing 1.3 mg/mL of p-nitrophenyl-*N*-acethyl-b-d-glucosamine (Sigma Aldrich) in 0.1 M citrate solution (pH 4.5) were added, and plates incubated for 30 min at 37 ◦C. Reactions were stopped by addition of 100 μL of 0.2 M glycine solution (pH 10.7), and OD at 405 nm was determined. The extent of secretion was expressed as the percentage of the total beta-hexosaminidase activity in the wells discounted of the values obtained in the supernatant of unstimulated cells [17]. Beta-hexosaminidase activity results are shown as mean ± s.d. of three independent experiments.
