Fig. 3. Flow cytometry analysis of pasteurized A. muciniphila binding to glycoengineered HEK293 cells.

a Schematic depiction of the gating strategy (GFP-positive/negative) for assessing binding to glycans on HEK293WT cells and on the mucin-GFP reporters expressed on a subpopulation of HEK293 cells using transient transfections. Live cells were gated on the side scatter area (SSC-A) versus forward scatter area (FSC-A) plot followed by gating on GFP positive cells (expressing mucin-GFP reporters) or GFP negative (not expressing mucin-GFP reporters). b Binding of A. muciniphila (0.625–7.5 × 10⁸ CFU/mL) to HEK293 cells glycoengineered as indicated without transfection of GFP-mucin reporters. Cells were analyzed with (+) and without (−) pretreatment with Clostridium perfringens neuraminidase (Neu, 10 mU, 1 h). Data points represent the average median fluorescence intensity (MFI) of two biological replicates. c–e Binding of A. muciniphila to glycoengineered HEK293 cells transiently transfected with GFP-tagged mucin reporters (MUC1, MUC2) using the GFP-gating strategy. c Binding to the GFP-negative cell population not expressing the mucin reporters reproduce binding found in b. Binding the GFP-positive cell population expressing the mucin reporters (cells) is shown in d and in e after subtraction of binding to the GFP-negative cell population. Note, expression of the MUC1 reporter did not significantly contribute to the binding of A. muciniphila, while expression of the MUC2 reporter selectively enhanced binding only in cells glycoengineered for core3 O-glycosylation (also note that neuraminidase pretreatment did not significantly affect this binding). One representative experiment is shown of n = 2 biological replicates. Source data are provided as a Source Data file.