Seven amino actinomycin d

Colonic LP cells were incubated with anti-mouse CD16/CD32 (BioLegend) to block non-specific Fc receptors, followed by a cell surface staining with the corresponding mixture of fluorescently-labelled monoclonal antibodies. Seven-amino actinomycin D (BioLegend) was used to discriminate live and dead cells. The following antibodies conjugated with biotin, PE, peridinin chlorophyll-Cy5.5, PE-Cy7, APC, APC-Cy7, brilliant violet (BV) 421, BV510, BV650, BV711, or BV785 were used for flow cytometry: anti-B220, anti-CD4, anti-IA/IE, anti-Ly6G, anti-Siglec F (all from BD Biosciences), anti-CD3e, anti-CD8a, anti-CD11b, anti-CD11c, anti-CD45, anti-CD45.2, anti-CXCR4, anti-F4/80, anti-TER119 (all from BioLegend), anti-Ly6C, anti-NK-1.1, anti-Siglec F (all from Miltenyi Biotec, Auburn, CA) and anti-CCR2 (R&D Systems). Following extracellular staining, the cells were fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences) and sequentially incubated with rabbit anti-collagen type I (Rockland, Limerick, PA), followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen). Data were acquired on LSRFortessa and processed using FlowJo software (Tree Star, Ashland, OR) with appropriate isotype controls to determine gating. Cell sorting was performed using FACSAria II (BD Biosciences).

Isolated cells were incubated with anti-mouse CD16/CD32 (BioLegend) to block non-specific Fc receptors. Then, the cell surface was stained with the corresponding mixture of fluorescently labelled monoclonal antibodies against B220, CD3, CD11b, CD45, F4/80, MHC II (IA/IE), Ly6C, Ly6G, Siglec F (BioLegend), NK-1.1 (Miltenyi Biotec, Auburn, CA, USA) and CCR2. The lineage cocktail consisted of antibodies targeting B220, CD3, Ly6G, NK1.1 and Siglec F. Seven-amino actinomycin D (BioLegend) was used to discriminate live and dead cells. Isolated cells were first gated on size, singularity and positive expression of EGFP and CD45. Next, lineage- and Seven-amino actinomycin D-positive cells were eliminated. To stain the intracellular antigens after surface labelling, cells were fixed and permeabilised with Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA, USA) and sequentially incubated with rabbit anti-collagen type I (Rockland, Limerick, PA, USA) and Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen). Data were acquired on LSRFortessa (BD Biosciences) and processed using FlowJo software (Tree Star, Ashland, OR, USA) with the appropriate isotype controls to determine the gating.