Erythrocyte Glycation Model Protocol

Erythrocytes were obtained from the French blood national agency (EFS-LR agreement number # 2018001378). Blood was collected in EDTA tubes (BD vacutainer^®, Franklin Lakes, NJ, USA). Erythrocytes were washed 3 times with an isotonic Ringer-lactate solution (B. Braun, Melsungen, Germany) and prepared at 20% hematocrit in phosphate buffered saline solution/5 mM glucose (PBS/0.1% glucose). Glycation was induced by incubating erythrocytes in the absence (G0) or presence of D-glucose at three increasing concentrations (5 (G5), 50 (G50) and 100 mM (G100)). A previous study, performed in our laboratory, showed that such in vitro conditions represent a suitable erythrocyte glycation model, inducing HbA1c percentages similar to those that can be measured in diabetic patients [16 (link)]. After 5 days of incubation at 37 °C, erythrocytes were washed 3 times with Ringer-lactate solution before a direct use by FACS, ektacytometry and for endothelial cells stimulation. Quantitative determination of hemoglobin in erythrocytes was performed with Drabkin’s colorimetric assay.