Ndfip1-cKO mice have been described 
. All experiments described in this manuscript compare Ndfip1-cKO mice to littermate controls. These controls include mice with one or two Ndfip1 floxed alleles but not expressing Cre, or mice expressing Cre that do not have either Ndfip1 allele floxed. No differences were observed when comparing these two types of controls. All mice were bred in the Children's Hospital of Philadelphia animal facility. All experimentation was approved and followed guidelines established by the institutional animal care and use committee of the Children's Hospital of Philadelphia.
Ndfip1-cKO mice were genotyped using purified genomic DNA from tail samples and the following PCR primers: Ndfip1 floxed forward 5′-TGAGGAAACAGACACACAATG-3′, Ndfip1 floxed reverse 5′- TGGAATGAACCTGAGGTCTCC-3′. Samples were amplified using conventional PCR techniques and run by electrophoresis on a 1% agarose gel. Wild type DNA forms an approximately 1 kb band and Ndfip1−/− DNA yields an approximately 340 bp band.
Animals were provided with autoclaved drinking water supplemented with sucralose (1.5 mgmL−1) or autoclaved drinking water supplemented with sucralose (1.5 mgml−1), ampicillin (0.5 mgml−1), gentamicin (0.5 mgml−1), metronidazole (0.5 mgml−1), neomycin (0.5 mgml−1) and vancomycin (0.5 mgml−1). For mice treated upon weaning, animals were 26 days old at the start of treatment and were sacrificed 14 days later. For mice treated since birth, pregnant females were treated starting approximately 1 day before giving birth until their litters were weaned, at which point weaned pups were treated for a further 14 days and sacrificed.
Lymphocyte isolation from tissues
At necropsy, mesenteric lymph nodes (mLN), spleen, esophagus and small bowel were harvested. Single-cell suspensions of spleen and mLN were prepared by mashing tissue through a 70 µm filter. LNs were then washed and resuspended in phosphate-buffered saline (PBS). Splenic red blood cells were lysed with ACK buffer (.15 M Nh4Cl, 10 mM KHCO3, .1 mM EDTA) and samples were then washed and resuspended in PBS.
For preparation of lymphocytes from the small bowel, a 3–4 inch section of small bowel was removed. Peyer's patches were excised and the luminal contents were removed.
Small bowel and esophagus samples were minced and then digested in medium containing collagenase type 1, collagenase type 1a, and DNAse for approximately one hour at room temperature. Samples were then strained through a 100 µm filter, washed with Dulbecco's Modified Eagle Media (DMEM), and filtered again through a 40 µm filter.
Single-cell suspensions of lymphocytes isolated from tissues were stained for surface expression using fluorochrome conjugated anti-CD3, anti-CD8α, anti-CD8β, anti-CD4, anti-F4/80, anti-CD62L, anti-CD44, and anti-Siglec-F (anti-Siglec-F was purchased from BD Biosciences; all other antibodies were purchased from Biolegend). Cells were stained for 30 minutes on ice and washed twice with PBS containing fetal calf serum and sodium azide (FACS buffer). Flow cytometry was performed on an LSRFortessa flow cytometer (BD Biosciences) and results were analyzed using Flow-Jo software (Treestar USA).
At necropsy, small bowel and esophagus sections were removed, fixed in 10% formalin for 48 hours, and embedded in paraffin. Sections were stained with eosin and hematoxyin. Stained sections were analyzed using a Leica microscope (Bannockburn, IL) with a bright field objective.
Stool samples were collected on day 0 of the experiment prior to sucralose or antibiotic administration and on day 13 (for mice treated upon weaning) or day 34 (for mice treated since birth). Samples were stored at −80 C and later defrosted and lysed by bead beating using the Mini BeadBeater-16 (Biospec). The samples were then prepared using the QiaAmp DNA Stool Mini Kit (Qiagen) according to the manufacturer's instructions. Final DNA concentrations were measured using a Thermo Nanodrop 2000.
Bacterial quantitation by 16S qPCR
16S quantitation was performed using real-time PCR of 20 uL triplicate reactions containing 10 ng stool DNA, TaqMan Environmental Master Mix 2.0 (Applied Biosystems), and primers/probe specific for bacterial 16S rDNA as described by Hill, et al.
. Standard curves with a range of 101
copies of E. coli
16S rDNA were prepared using linearized plasmid containing a single copy of the 16S gene.