Isolation of Jejunal Lamina Propria Lymphocytes

Lamina propria lymphocytes (LPLs) from the jejunum were isolated as reported by us earlier [28 (link),30 (link)]. In brief, jejunum biopsies were obtained by endoscopy, washed properly in chilled sterile PBS, minced with scissors, and digested with EDTA solution in a 37 °C shaker for 30 min to detach the majority of intestinal epithelial cells. After the removal of epithelial cells by filtration through screen cup strainer with 50 mesh size (0.229 mm) (Sigma-Aldrich, St. Louis, MO, USA), the remaining tissues on the strainer were scrapped and further minced with scissors and digested with media containing 60 U/mL of collagenase (type II, Sigma-Aldrich). The cell suspension was passed through a 16-gauge feeding needle for better separation of any clumps. The larger clumps were removed using a nylon biopsy bag (ThermoFisher, Waltham, MA, USA). To enrich LPLs, cells were centrifuged over discontinuous percoll (Sigma-Aldrich) gradients. The interface layer containing LPLs between the two percoll layers was collected, washed in PBS, and resuspended with complete media. This percoll separation removed dead cells and enriched the cell population for lymphocytes. However, these enriched LPLs also contain trace amount of epithelial and other leukocyte positive cells as reported by us elsewhere [31 (link)]. The enriched LPLs were used for flow cytometry assays.