Anti fcγr antibody clone 24g2

Caecum and proximal colon were excised and intra epithelial lymphocytes (IEL) and lamina propria lymphocytes (LILP) were prepared essentially as described [52 (link)] with slight ±modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) and processed with ebioscience fix/perm buffer as per manufacturer’s instructions before labelling with antibodies specific for CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience) and T-bet (clone TWAJ; eBioscience). All samples were analysed on a FACS LSRII.

Spleens and mesenteric lymph nodes (mLNs) were removed from mice and disaggregated through a 100 μm sieve. Small intestines were excised and lamina propria lymphocytes (SILP) were prepared essentially as described [80 (link)] with slight modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30 min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) before labelling with antibodies specific for CD3 (eBio500A2), CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience), IL-13 (clone eBiol13A; eBioscience), IFNγ (clone XMG1.2; eBioscience), IL-17(eBio17B7; eBioscience), IL-9 (RM9A4e; Biolegend) or p-Smad 2/3 (Santa Cruz). For intracellular cytokine analysis cells were incubated for 12 hours with 1x Cell stimulation cocktail (plus protein inhibitors) (ebioscience). Cells were then stained with antibodies using the eBioscience Foxp3 permibilization kit according to the manufacturer's instructions. For pSmad2/3 staining, an Alexa Fluor 594-labelled donkey anti-goat secondary antibody was used (Invitrogen). All samples were analysed on a FACS LSRII.