Validation of Gpr44 ^fl/fl -GFP mice and determination of ILC3s within the lamina propria. (A) Schematic of mouse design. LoxP sites were inserted into the Gpr44 exon 3, followed by the sequence encoding eGFP. The presence of Cre recombinase excises the targeted segment of Gpr44 exon 3, resulting in deletion and allowing for transcription of eGFP from the endogenous Gpr44 promoter. (B) Genotyping PCR showing cell lineage-specific deletion of the targeted Gpr44 exon 3 sequence. Recombination occurs only in the presence of Cre in a cell lineage-specific manner in small intestinal (SI) epithelial cells (IECs) but not splenocytes in Vil1-Cre Gpr44^fl/fl-GPF mice. WT allele = 239 bp; Flox allele = 348 bp; Flox allele after recombination = 279 bp. (C and D) Representative images of duodenum, jejunum, and cecum sections from naive Gpr44^fl/+Rorγt-Cre⁺ mice showing cells positive for DCLK-1 (white), CRTH2-GFP (green), and CD4 (red); blue color indicates DAPI counterstained nuclei. Scale bar = 50 µm (C); representative image (D) quantification of CD4⁺ cells or CRTH2⁺ cells from nearest tuft cells. n = 3 mice; ns = not significant. (E) Lamina propria cells were isolated from Gpr44^fl/flRorγt-Cre or Gpr44^fl/+Rorγt-Cre and stained with a flow panel as described in Materials and methods to identify ILC3s. To analyze the ILC3s, CD45⁺ cells were gated and lineage negative (CD3⁻, CD19⁻, and CD14⁻) cells were classified into two groups: GFP high and GFP low. Both groups were then further gated into CD90⁺, CD127⁺, NK1.1⁻, KLRG1⁻, and Rorγt⁺. Then they were further analyzed for the presence or absence of CCR6 and NKp46. (F) Quantification of different subtypes of ILC3s out of total GFP (CRTH2)-expressing cells. Source data are available for this figure: SourceData FS3.