2.9.1. Isolation of Human Erythrocytes

Venous human blood was collected from randomized patients of Cova da Beira Hospital Centre (Covilhã, Portugal) by antecubital venipuncture into K₃EDTA vacuum tubes, in accordance with the protocol approved by the Health Ethical Commission of Cova da Beira Hospital Centre (no. 25/2018) without the need for written informed consent of the patients. Therefore, erythrocytes from blood collected in the early morning already processed by the hospital and without any need for more studies. Then, they were isolated according to Gonçalves et al. [1]. Briefly, 4 mL of a collected sample was transferred to sterile conic tubes, mixed with 6 mL PBS at pH 7.4 and centrifuged at 1500× g for 5 min at 4 °C. This procedure was repeated three more times, and the resulting supernatant from each centrifugation was always discarded. The number of cells (cells/mL) and viability (always above 98%) were obtained by the Trypan blue exclusion method. The suspensions of isolated erythrocytes were kept on ice until use.