Enzymatic Exofucosylation of Murine Pericytes

Murine pericytes were modified by enzymatic exofucosylation as previously reported (Garcia-Bernal et al., 2020 (link)). Briefly, cells were resuspended at 2 × 10⁷ cells/ml in fucosyltransferase VII (FTVII) reaction buffer composed of Hanks Balanced Salt Solution (HBSS, Gibco) containing 30 μg/ml fucosyltransferase VII (FTVII, R&D Systems), 20 mM HEPES (Thermo Fisher Scientific), 0.1% human serum albumin (Grifols) and 1 mM guanosine 5′-diphospho-β-L-fucose sodium salt (GDP-fucose, Sigma Aldrich), and incubated for 60 min at 37°C and 5% CO₂. Unmodified controls pericytes were treated only with GDP-fucose (w/o FTVII) in the same conditions as above. Cell viability after exofucosylation was assessed by trypan blue exclusion (usually 95% live cells). Efficacy of exofucosylation was evaluated by analysis of HECA452 antibody (BD Biosciences) staining and calcium-dependent mouse E-selectin/human IgG chimera (R&D Systems) binding by flow cytometry.