Discontinuous percoll gradient

For most analyses involving APC subsets, lymphoid organs were injected and digested with Liberase TL (400 μg/mL, Roche) and DNase (800 μg/mL, Roche) in RPMI for 30 min at 37°C. When isolating APCs from the thymus, EDTA (10 mM) was added to digests and enriched by layering digested thymocytes on top of a discontinuous Percoll gradient (GE Healthcare) at 1.115 g/mL in PBS, followed by centrifugation at 1350 g for 30 min and isolation of cells settling at the Percoll interface (Aschenbrenner et al., 2007 (link)).

MLN, Peyer’s patches, intraepithelial lymphocytes (iEL) and lamina propria lymphocytes (LPL) were isolated by the method of Lefrancois and Lycke ³⁷ . Briefly, MLN were isolated and then the intestine from the duodenum to the rectum was flushed with Ca²⁺Mg²⁺-free 100 mM HBSS (CMF) (Sigma-Aldrich, St. Louis, MO). The colons were cleaned by removal of fat, fecal matter and debris. RPMI was injected into the intestine to highlight the Peyer’s patches, which were then collected with a fine forceps and scissors. The intestines were opened longitudinally and cut into 0.5 cm sections, shook twice with CMF solution containing 10 mM HEPES, 25 mM NaHCO₃, 1 mM DTT and 1 mM EDTA all from Sigma-Aldrich, St. Louis, MO, and 2% fetal bovine serum (FBS) Gibco®, Portland, OR at 37 °C. IELs were then purified using discontinuous Percoll gradients (GE Healthcare, Buckinghamshire, UK) by collecting mononuclear cells at the interface between the 40% and 70% layers. For the isolation of LPLs, intestinal pieces were digested for 70 min with complete RPMI-1640 containing 1 mg/ml collagenase (Roche Applied Science, Upper Bavaria, Germany), 40 μl/ml dispase (Sigma-Aldrich, St. Louis, MO) and 4 μl/ml Dnase I (Sigma-Aldrich, St. Louis, MO) at 37 °C. LPLs were then purified using 40%/70% discontinuous Percoll gradients.