eGFP+ sorted cells represent a highly enriched population of I-cells.

<p>A: Analysis of RNA extracted from eGFP+ and eGFP− cells. RNA was analyzed using a RNA 6000 PicoChip kit. Intact 28S and 18S ribosomal bands (gels, left panel) or distinct 28S and 18S RNA ribosomal peaks (electropherograms, right panel) confirmed integrity of extracted RNA from sorted cells. L indicates Pico 6000 RNA ladder that spans 0.2–6.0 kb. B: eGFP+ cells were highly enriched in Cck mRNA transcripts. Semi-quantitative PCR for Cck mRNA demonstrated that only eGFP+ cells contain Cck mRNA transcript, a marker of I-cells. Akp3 mRNA transcript, a marker of enterocytes, was mainly detected on eGFP− cells, showing little contamination of eGFP+ cells (I-cells) with enterocytes. 18S rRNA was used as a loading control. L declares Hyperladder IV (100–1000 bp, Bioline, UK).</p

I-cells contain GPR40, GPR120, GPR41, GPR43, GPR119 and CB1 mRNA transcripts.

<p>Semi-quantitative RT-PCR analysis revealed that eGFP+ cells (I-cells) were enriched in mRNA transcripts encoding Gpr40/Ffar1, Gpr41/Ffar3, Gpr43/FFAR2, Gpr119 and CB1 whereas Gpr120/O3far1 was enriched in eGFP+ cells, but was also present eGFP− cells; RT-PCR of 18S rRNA confirms that equal amount of cDNA template from eGFP+ and eGFP− cells was used for the analysis. L declares Hyperladder IV (100–1000 bp, Bioline, UK). Asterisk indicates bands representing primer dimers.</p

Semi-quantitative RT-PCR on amplified cDNA from sorted cells validates I-cells enriched mRNA transcripts.

<p>Semi-quantitative RT-PCR analysis using amplified cDNA from eGFP+ and eGFP− cells. Left panel: eGFP+ cells were highly enriched in mRNA transcripts encoding eGFP and CCK whereas these transcripts were not detected in eGFP− cells. eGFP− cells were enriched in mRNA transcripts encoding AKP3 and MUC2, markers of enterocytes and goblet cells respectively. Right panel: Validation of Gpr40/Ffar1, Gpr120/O3far1, Gpr41/Ffar3, Gpr43/Ffar2, Gpr119 and CB1 mRNA transcripts enrichment in I-cells. 18S rRNA was used as a loading control. PCR products were amplified simultaneously, using different number of cycles (low-for lower number of cycles and high-for higher number of cycles). Equal volumes of each reaction were run on the same 2% agarose gel. L declares Hyperladder V (50–250 bp, Bioline, UK). Asterisk indicates primer dimers.</p

eGFP expressing cells are strongly immunopositive for CCK.

<p>Representive images of transverse sections of duodenum from CCK-eGFP mice. A: eGFP-tagged cells (green) were flask-shaped with a diffuse distribution, characteristic of ‘open-type’ enteroendocrine cell. B: Immunostaining with anti-CCK antibody (red) showed colocalization of CCK and eGFP in ∼ 90% of eGFP-labelled cells. C: High magnification micrograph (2.5X digital zoom) revealed I-cells had a narrow apical membrane (arrow) that was in contact with gut lumen and a broad basolateral membrane (asterisk) with adjacent strong CCK staining. In all images, nuclei were counter-stained with Hoechst 33342 (blue).</p

FACS-sorting of eGFP+ and eGFP− cells.

<p> A: Schematic overview of the methods employed to isolate I-cells and perform transcript analysis. B: Dissociated duodenal cells from eGFP-CCK mice were sorted using a BD FACS Aria cell sorter (lower panels). Dissociated duodenal cells from a WT/CD-1 mouse were used as a control to set gating parameters for analysis and sorting (upper panels). Cells were first stained with PI and analyzed by eGFP (X-axis) and PI (Y-axis) fluorescence intensity (left panels). PI- positive cells (dead cells, P0) were excluded from further analysis. Live cells (Blue, P1) from CCK-eGFP mice displayed a population of highly fluorescent cells (purple rectangle) that were not present in cell from WT mice. Live cells were also analyzed based on eGFP fluorescence intensity (X-axis) and side scatter (Y-axis). Cells that expressed eGFP were gated as eGFP+ cells, representing 0.5% of total cell population (P2, right lower panel). This population was not present in cells from WT mice (P2, right upper panel). All other cells (P3) were gated as eGFP−. eGFP+ cells and approximately equal number of eGFP− cells were sorted.</p