Flow cytometry detection of Neu5Gc and erythrocyte receptors

Neu5Gc and erythrocyte receptor surface expression was assessed by flow cytometry. Cells were washed three times in PBS containing 0.5% Neu5Gc-free blocking agent (Siamab) and pelleted at 500 g for 4 min in 96-well plates at 5 × 10⁵ cells/well. Cells were incubated with antibodies at the following dilutions: anti-Neu5Gc (1:5000, Siamab), phycoerythrin(PE)-conjugated anti-DARC (1:10, 130-105-683, Miltenyi Biotec), anti-CD71-PE (1:10, 130-104-151, Miltenyi Biotec), anti-BSG (1:1000, Clone MEM-M6/6, Axxora [Exbio]), fluorescein isothiocyanate-conjugated anti-glycophorin A (GPA) (1:50, Clone 2B7, 60152FI, STEMCELL Technologies) and anti-glycophorin C (GPC)-FITC (1:500, BRIC 10, sc-59183 FITC,Santa Cruz). After 1 h incubation at room temperature and three washes in blocking buffer, unstained cells and cells stained with DARC, CD71, GPA and GPC antibodies were resuspended in 100 µl PBS for flow cytometry analysis (MACSQuant; Miltenyi Biotec). Neu5Gc-stained cells were incubated in anti-chicken IgY-Alexa Fluor 488 secondary antibody (Life Technologies) at 1:1000 for 30 min at room temperature. Unstained control samples were incubated in anti-mouse IgG2a-PE (1:10, Miltenyi Biotec), anti-chicken IgY-Alexa Fluor 488 antibody or anti-mouse IgG-Alexa Fluor 488 (1:1000, Life Technologies). Cells were washed twice before flow cytometry. Data were analysed in FlowJo 4 version 10.0.7 (Tree Star).