Quantifying Cell Surface Glycans by Flow Cytometry

Flow cytometry was carried out according to standard methods as described in [19 (link)] using an Attune NxT flow cytometer (ThermoFisher Scientific). Cells were detached with non-enzymatic cell dissociation solution (Merck) or briefly (for Itgb4 staining only) trypsinised with 0.04% trypsin/ 0.03% EDTA (PromoCell). For surface GAG detection, cells were treated with 1mU of heparinase III (EC4.2.2.8 from Flavobacterium heparinum) to expose neo-epitope of heparan sulphate or with chondroitinase ABC (EC 4.2.2.4 from Proteus vulgaris) (AMS Biotechnology) for 1h at 37°C prior to the staining procedures. Antibodies were Δ-HS (F69-3G10, AMS Biotechnology), CS (CS56, Merck), perlecan (7B5, ThermoFisher Scientific), glypican-1 (AF4519), integrin β4/ CD104 (clone 439-9B, eBioscience), integrin β1/ CD29 (P4C10, NBP2-36561), syndecan-2 (MAB2965), biglycan (AF2667), laminin α5 (NBP2-42391) from Biotechne. Isotype control mouse IgG1 (P3.6.2.8.1; 14-4714-81 from Invitrogen), mouse IgG2b (MG2B00), goat IgG (AB-108-C from R&D), rat IgG2b (14-4031-81), mouse IgM (PFR-03) and fluorophore-conjugated secondary antibodies goat anti-mouse IgG PE (12-4010-82), donkey anti-goat IgG FITC (A16000) and anti-rat IgG FITC (31629) were from ThermoFisher Scientific. The main population was gated by forward and side scatter plot of untreated cells using FlowJo (v9); among this, single cell population of 10⁴ cells per condition was subjected to analysis. Mean fluorescence intensity was determined and presented as % relative to untreated control.