Supplementary Figure 2: Generation and analysis of IL-9–citrine reporter mice

(a) Generation of II9^Cit reporter mice. The targeting strategy to insert the citrine reporter gene at the start codon of the II9 gene is shown. neo, neomycin cassette; TK, thymidine kinase cassette; e1, II9 exon 1. The black rectangle represents the 5' probe for Southern blot screening. (b) Southern hybridization for II9^Cit-targeted clones. A positive colony of an II9^Cit embryonic stem cell clone using the 5' probe is shown. Wild-type allele, 7.0 kb; targeted allele, 6.0 kb. (c) Detection of II9 in CD4⁺ T cells differentiated in vitro using citrine reporter (citrine - top panels) or intracellular staining (ICS – lower panels). Before FACS analysis CD4⁺ T cells were purified from naïve spleen cells and plated under the following conditions for 72 hours. T_H0: plate bound anti-CD3 and anti-CD28 antibodies. T_H1: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL2, IL12, anti-IL4 antibodies. T_H2: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL2, IL4, anti-IFN-γ antibodies. T_H9: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL4, TGF-β. (d) Detection of II9^Cit and IL9 ICS in mice following rIL33 administration. Mice were treated with three daily doses of IL33. Mesenteric lymph nodes (MLN) were harvested and cells were analyzed for citrine expression or IL9 ICS. Representative of three biological replicates are shown. (e) Detection of Il9^Cit following Nippostrongylus brasiliensis infection. Six days post-infection MLN were harvested and analyzed for citrine expression. Only ILC2 in the mesenteric lymph nodes on day 6 expressed citrine. T cells and other cell types in other organs and other time points did not express detectable citrine at this time point (not shown). Representative images of three biological replicates are shown.