Isolation of Colonic Lamina Propria Cells

Isolation of colonic LP cells was performed as previously described [18 (link)]. Briefly, colonic tissue was cut into pieces 0.5 cm in length and placed in an orbital shaker for 20 min at 37 °C in HBSS with 10% FCS and 2 mM EDTA. This shaking step was repeated and then the remaining tissue was minced and placed in HBSS with 10% FCS, 1 mg/mL collagenase IV (Sigma), and 40 U/mL DNase (Roche) I and shaken for 20 min at 37 °C. The contents are then filtrated through a 70 μm cell strainer directly into a 50 mL conical tube. Each 50 mL conical tube is topped off with HBSS/FBS and centrifuged at 4 °C. After the repetition of this step, the supernatant is poured off and the cell pellet is resuspended in ice-cold HBSS/FBS. From these resulting LP cells, CD45+ immune cells were isolated by using anti-CD45 magnetic beads (Miltenyi) and subsequently used for downstream analysis.