Coomassie brilliant blue g 250

To confirm survivin recombination, surv.VLP were run on a 10% SDS-PAGE gel and visualized by Coomassie Brilliant Blue G-250 staining (BD Biosciences, San Jose, CA, USA). The resulting band was excised and the survivin epitope was identified by mass spectrometry using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF, Centre for Protein Research, University of Otago, Dunedin, New Zealand).

Dimethyl sulfoxide (DMSO), TRIS, sodium hydrogen phosphate (Na₂HPO₄), and sodium acetate (CH₃COONa) were from Merck (Darmstadt, Germany). Ethylenediaminetetraacetic acid was from Fluka (Buchs, Switzerland). Acrylamide, Coomassie Brilliant Blue G-250 and Triton X-100 were from BDH (Poole, UK). Hepes, Brij-35, Silver nitrate, alkaline phosphatase-conjugated antibodies and gelatin were from Sigma (St. Louis, MO, USA). gelatin-Sepharose, was from GE-Healthcare (Uppsala, Sweden). DC Protein Assay and unlabeled molecular weight standards were from BioRad (Richmond, CA, USA). Sf9 insect cells and Magic Marker molecular weight standards were from Invitrogen (Carlsbad, CA, USA). Western Blotting Luminol reagent and HRP-conjugated donkey anti-goat secondary antibody were from Sancta Cruz (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit secondary antibody was from Southern Biotech (Birmingham, AL, USA). Fetal bovine serum was from Biochrom AG (Berlin, Germany). Galardin (Gm6001), human MT1-MMP/MMP-14 (catalytic domain), TLN and PLN were from Calbiochem (San Diego, CA, USA) and Aureolysin was from BioCentrum Ltd. (Kraków, Poland). McaPLGL(Dpa)AR-NH₂ (ES001) and McaRPPGFSAFK(Dnp)-OH (ES005) were from R&D Systems (Minneapolis, MN, USA).

Bicinchoninic acid (BCA) protein assay kit for low concentrations (ab207002, Abcam) and Bradford assays (in-house made using Coomassie Brilliant Blue G-250, 44,329, BDH) were used to quantify the protein content of SS-hAFSC-conditioned medium and sEV isolates. BCA assays were performed according to the manufacturer’s instructions (300-µl final reaction volumes; 2-h incubation time). Higher protein content samples were analysed using standard BCA assay (Sigma) in 200-µl final reaction volumes for a 30-min incubation time. Absorbance for both assays was read at 562 nm on an FLUOstar plate reader (BMG Labtech) and protein concentrations were calculated using BSA standards and a four-parameter logistic curve.
Bradford assays were performed in 300-µl final reaction volumes, with 10-min incubation time and measurement of absorbance at 595 nm on an FLUOstar plate reader (BMG Labtech). Protein concentrations were calculated using BSA standards and a four-parameter logistic curve.