*5.4. Cell Treatment with B. jararaca Venom*

In a six-well plate, 2 × <sup>10</sup><sup>5</sup> cells were plated and after the cells had reached a confluence of 70–80% (2–3 days), different concentrations of *B. jararaca* venom were added to the culture medium, starting from the cell cytotoxicity threshold up to a 1000-fold dilution for 24 h. For mass spectrometry analysis, cells were washed with ice-cold PBS and lysed with 1 mL ice-cold 8 M urea supplemented with cOmpleteTM protease and phosphatase inhibitors cocktail (Merck Millipore, Burlington, MA, USA). Fifty microliters of the cell lysate were separated for protein quantification using BCA (Bicinchoninic acid assay, Thermo Pierce, Walthan, MA, USA) and the lysate were stocked at −80 ◦C until further preparation of the sample for mass spectrometry analysis. Both cytotoxicity and proteomics experiments were performed in three biological replicates.
