Intestinal Epithelial and Lamina Propria Isolation

Intestinal samples (6–10 cm long pieces of jejunum and colon) were collected in 50 ml tubes with ice-cold calcium and magnesium free HBSS (Fisher Scientific) and immediately transported to the laboratory. Fat, blood vessels, and mesenteric lymph nodes were trimmed. Mucus and gross debris were quickly removed by covering the specimen with dry paper towels. Specimens were further washed twice in cold PBS (pH 7.4). Intestinal samples were cut into small pieces (approximately 0.5–1 cm²).
All isolation protocols were performed on tissues from necropsied animals to maintain consistency of protocols compared to biopsy protocols where techniques may result in different proportions of ECs in samples. In brief, tissues were treated in 2 major ways. In one technique, the intestinal EC were separated from intestinal pieces by incubating 0.5–1 cm² pieces of tissue in Dithriothreitol (0.15%, DTT, EMD Chemicals) (Fig. 1) [21] (link) followed by EDTA with shaking at 37°C. In another protocol, minced tissues were directly treated with EDTA solution (Fig. 2) as reported earlier [53] (link), [81] (link). Mucus and large debris were removed from the supernatant by filtering through loosely packed glass wool. After epithelial removal, LPL were collected by mincing the remaining tissue into 1–2 mm pieces, followed by digestion in complete RPMI-5 medium containing 5% fetal calf serum (FCS) (RPMI-5) containing 60 units/ml of Type II collagenase (Sigma-Aldrich) again with shaking at 37°C. For enrichment of lymphocytes, supernatants of LPLs were centrifuged over discontinuous Percoll (Sigma-Aldrich) density gradients followed by washing with PBS [53] (link), [81] (link). All isolated cells were washed twice and resuspended in complete RPMI-10 medium containing 10% FCS before staining. All cells were >90% viable by trypan blue dye exclusion method.