Isolation of Intestinal Lamina Propria Macrophages from Septic Mice

Lamina propria macrophages from the intestinal mucosa of septic mice were isolated as previously reported^{49 (link), 50 (link)}. Briefly, the tissues were first predigested to eliminate epithelial cells. The intestinal tissues were then digested with digestion solution (1 mg/mL collagenase type VIII + 3 mg/mL dispase II + 40 μg/mL DNase I + 5% FBS) + Hanks Solution (all reagents were from Sigma-Aldrich, St Louis, MO, USA). After incubation at 37 °C for 30 min while shaking (150 rpm), the tissues were filtered through a 100-μm nylon mesh filter (Fisher brand, Loughborough, UK). The remaining pieces were repeatedly digested and filtered, and the filtered suspensions were rinsed with PBS. The cell suspensions were centrifuged, resuspended in fresh PBS, and filtered again through a 40-μm nylon mesh filter (Fisher brand, Loughborough, UK).