Apis mellifera

A volume of 50 μL BV (0.1 mg lyophilized whole venom of Apis mellifera (Sigma Co., St. Louis, USA) dissolved in 0.9% sterile normal saline (NS)) was intraplantarly injected into right hindpaw to produce nociception [19 (link)]. A concentration of 20 μM octreotide diluted in 0.9% NS (Sandostatin, Novartis Pharma Schweiz AG, Bern, Switzerland) was utilized in the present study based on the literature [6 (link)].
SD rats were divided into five groups: (1) NS + NS group: 60 μL NS intraplantar injection followed by 50 μL NS injection as the control group; (2) NS + BV group: 60 μL NS intraplantar injection followed by 50 μL BV injection as the pain model group; (3) ipsilateral OCT + BV group: 60 μL octreotide (20 μM) injection prior to the BV injection; (4) contralateral OCT + ipsilateral BV group: 50 μL BV injection into the right hindpaw after injection of 60 μL octreotide into the contralateral hindpaw to determine whether the local injection of octreotide can bring about systemic effects; (5) c-SOM + OCT + BV group: local injection of 60 μL, 128 μM cyclosomatostatin (c-SOM, Sigma Co., St. Louis, USA), a somatostatin receptor (SSTR) antagonist, before the intraplantar injection of 60 μL octreotide to determine whether the effect of octreotide was somatostatin receptor specific. A ten-minute interval was allowed between the applications of each drug in all groups.

Enzyme treatments were performed on 4-day old biofilms. Planktonic cells were removed and biofilms were washed once with PBS prior to adding 150 μL of enzyme, or the corresponding buffer without enzyme as control. After 20 h of incubation at 37°C, planktonic cells were removed and the biofilm washed once with 150 μL PBS prior to quantification by crystal violet staining as described above. The following enzymes were used: TURBO^® DNase (Thermo Fisher Scientific) in the commercial buffer provided, Cellulase from Trichoderma sp. (Sigma) in 50 mM citrate buffer (pH 5.0), α-amylase from Bacillus licheniformis (Sigma) in PBS (pH 7.4), α-mannosidase from Canavalia ensiformis (Sigma) in 10 mM ammonium acetate buffer (pH 7.0), Proteinase K (GoldBio) in 10 mM Tris buffer (pH 8.0), Trypsin from bovine pancreas (Sigma) in PBS (pH 7.4), Lipase from Candida rugosa (Sigma) in PBS (pH 7.4), Lysozyme (Sigma) in PBS (pH 7.4) and phospholipases A1 (from Aspergillus oryzae; Sigma) and A2 (from Apis mellifera; Sigma) in 50 mM Tris–HCl (pH 7.5), 10 mM CaCl₂ and 2% DMSO buffer.

Immunoblotting was performed by separating 5 μg of the different recombinant proteins (rLlPLD1 and rLlPLD2 from L. laeta, LiSMDP1 from L. intermedia, LrSMD1 from L. reclusa, and LgDerProt1 from L. gaucho), or 5 μg of L. laeta and Sicarius venom, using a 12% SDS-PAGE gels under non-reducing conditions. Additionally, 5 μg of phospholipase A₂ (PLA₂) from bee venom (Apis mellifera) (Sigma-Aldrich Co, St Louis, MO, USA) and phospholipase C (PLC) from Bacillus cereus (Sigma-Aldrich, USA) were tested. Gels were stained with Coomassie Brilliant Blue or transferred to a nitrocellulose membrane. After transfer, the membranes were blocked for 2 h with 5% non-fat milk in TBS/0.1% Tween20 (TBS-T) and incubated for 1 h at room temperature with pooled sera from Groups 1 and 2 (1:1000 dilution) or with purified and immunoselected IgGs from both groups at 1 μg/mL. Membranes were washed six times for 10 min each with TBS-T and incubated with goat anti-human HRP-IgG antibody (1:50,000 dilution) in TBS-T for 1 h at room temperature. After another six washes with TBS-T, the membranes were developed with the ECL™ Western blotting detection reagent kit (GE Healthcare, Chicago, IL, USA).