Id cellstab

Human RBCs from different sources (AbtectcellTM III 3%, Commonwealth Serum Laboratory (CSL) and donor samples obtained from Australian Red Cross Blood Service diluted to 3% in ID CellStab, Bio-Rad Laboratories Pty. Ltd.) were prepared for 3 different conditions. 1) Blank controls were RBCs without any form of incubation. 2) RBCs for temperature control were incubated at 37 °C for 30 minutes without addition of antibodies. 3) RBCs for positive samples were D+ incubated at 37 °C for 30 minutes with 2% for further manufacturing use (FFMU) IgG anti-D for further manufacturing use (FFMU), CSL. Blood smears from the 3 samples were prepared on microscope glass slides and air dried. Glass slides were imaged with a JPK Nanowizard 3 AFM in AC (intermittent contact) mode using Bruker NCHV model cantilevers, which had nominal resonant frequencies of 320 kHz and spring constants of 42 N/m. Images were obtained with a set-point force of <1 nN. Scans of 1 μm × 1 μm on random regions of the red blood cell surface were performed for the RMS roughness study.

Blood from healthy volunteers and 3 OHSt patients with the Phe65Ser RhAG mutation was obtained by venipuncture on the same day ektacytometry was done. All samples containing the whole cells from blood were washed with NaCl 0.9% and frozen in 20% glycerol solution as previously described [39 (link)]. These samples were stored by the Centre National de Référence des Groupes Sanguins (CNRGS). To remove glycerol, the blood cells were first washed in glycerol 10%, then in sorbitol 8%, and finally three times in NaCl 0.9%. Finally, RBCs derived from the last pellet were resuspended in ID-CellStab (Biorad, Marnes la Coquette, France) and washed in DPBS (Dulbecco’s Phosphate Buffer Saline Gibco, ThermoFisher Scientific, Illkirch, France) before being used in some of the experiments described below.

100 μL EDTA-anticoagulated blood samples were washed with sodium chloride (0.9%, Fresenius Kabi Austria, Linz, Austria) at 79 g for 3 minutes. Erythrocytes were incubated for 30 minutes at 37 °C with antibodies in ID-CellStab (buffer specially formulated for erythrocytes; Bio-Rad Laboratories, Cressier, Switzerland). Unbound antibodies were removed by washing three times with sodium chloride. Subsequently, cells were resuspended in ID-CellStab.