Tnfα alexa 488

Leukocytes were isolated from the colon as described in the literature (Nguyen et al., 2015 (link)). The colons were cleansed in HBSS containing 2% BCS without Ca²⁺/Mg²⁺. The tissues were first incubated twice with HBSS containing 2% BCS 2 mM EDTA at 37°C for 20 minutes. The EDTA was then rinsed with HBSS containing 2% BCS without EDTA. The colon tissues were cut into ~0.5 cm pieces and, incubated twice with collagenase IV (Sigma Aldrich, St Louis, Mo) in 5% BCS RPMI at 37 °C (30 minutes each). The cell suspension was then collected and washed. Density gradient centrifugation with 40/80% percoll (GE Healthcare Bio Science AB, Uppsala, Sweden) was used to enrich the leukocytes at the interface. The leukocytes were then washed and stimulated with a mixture of LPS (1μg/mL) + PMA (50ng/mL) + Ionomycin (1 μg/mL) + Brefeldin A for 3 hours, before staining for flow cytometry analysis. The following dyes and antibodies were used to stain the cells: Zombie Aqua (Biolegend, San Diego, CA), CD45.2-PE/Dazzle 594 (Biolegend), CD45.1-Qdot 605 (Biolegend), IL17-Alexa 700 (Biolegend) and TNFα-Alexa 488 (Biolegend). Intracellular staining of cytokines was performed using Foxp3 fixation and permeabilization solutions per manufacturer recommendations (eBioscience). Data was acquired and analyzed on an LSRII (BD Biosciences) and FlowJo software (FlowJo, LLC), respectively.