2.5.1. Hemolytic Assay

Five mL of rabbit blood (previously extracted with citrate dextrose) were added to 45 mL of PBS. During 10 min, the samples were harvested by centrifugation (1000× g, 4 °C) and the supernatant was then discarded. This process was repeated two more times until the PBS solution was clear, thus indicating that the pellet was clean. PBS was then added to make a solution of 3% (w/V). Then, in each well of a 96-well plate, 80 µL of rabbit blood was deposited along with 80 µL of each sample. Three wells, each containing blood with PBS and Triton X-100 were used as negative and positive controls, respectively. During 4 h the plates were incubated at 37 °C, and afterwards centrifuged at 1000× g for 10 min, at 4 °C. Finally, the supernatant (80 µL) of each well was transferred into a 96-well-U-bottom cell culture plate and the absorbance at 558 nm (ABS) was measured. For each sample, data was normalized with the controls, using the following formula:

This research has been reviewed by the Animal Welfare Body of the School of Medicine and its Life and Health Science Research Institute (EM/ICVS), and the Research Institute on Biomaterials, Biodegradables and Biomimetics (I3Bs) - ORBEA EM/ICVS-I3Bs (Reference: ORBEA EM/ICVS-I3Bs_002/2019) as well as by the National Competent Authority Direção-Geral de Alimentação e Veterinária (DGAV) (Reference: 0421/000/000/2020 - 022434). The research has been approved by ORBEA on November 13th of 2019 and by DGAV on December 14th of 2020.