Anti il 17a efluor450

Strains (5 × 10⁸ CFU/day/mice) were administered by intragastric gavage to WT conventional BALB/c mice for 5 days. Colon samples were removed at sacrifice and stored in RNAlater® storage solution (Ambion, Life Technologies, Foster City, CA, USA) at − 80 °C until qRT-PCR analysis. MLN and intestine were harvested and immediately processed for flow cytometry. Cell suspensions of MLN and intestine (3–5 × 10⁶ cells, in RPMI1640 supplemented by 10% FCS, 2 mM l-glutamine, 2 mM HEPES, 40 mg/ml gentamycine) were stimulated using the Leukocyte Activation Cocktail containing BD GolgiPlug (BD Biosciences) (1 μl/ml of cell suspension) for 5 h. Cells were stained by mAbs anti-mouse CD11c eFluor450, CD11b eFluor 450, B220 eFluor 450, CD3 eFluor 450, CD117 Alexa Fluor 700, NK1.1 PerCP-Cy5.5, NKp46 FITC (provided by eBioscience), CD4 APC-H7, CD90.2 BV 500; CD45RB BV 605, MHCII BV 650 (provided by BD Bioscience, san Jose, CA, USA). Subsequently, cells were permeabilized and fixed using the Transcription Factor Buffer Set (BD Bioscience), and intracellular staining was performed using mAbs anti-IL-17A eFluor450, anti-FOXP3 PE-Cy7, anti-IL-22 PE and RORgt APC (eBioscience). Flow cytometry data were analyzed using software FlowJo. Gating strategy and representative dot plots for control and BIO5768 treated mice are presented in Figs. S1 and S2.