Mouse cd11c positive selection kit 2

Freshly isolated LI LPLs (2–3 × 10⁶) were cultured in RPMI with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Gibco, Life Technologies), with S. Typhimurium supernatant for 5 hours in total 500 μl culture volume. For the in vitro co-culture experiment using purified cells, ILC3s were sorted from LI LPLs as described above, and CD11c⁺ APCs were isolated from LI LPLs using the Mouse CD11c Positive Selection Kit II (STEMCELL, 18780A) after depletion of adaptive immune cells using PE positive Selection Kit II (STEMCELL, 17684). Cells were cultured for 5 hours and subjected to cytokine production examination for FACS analysis were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin for 4 hours unless indicated otherwise, and Brefeldin A (2 μg/ml) was added 2 hours before cells harvest. Cells used for qRT-PCR were directly collected after co-incubation with bacterial supernatant without PMA/ionomycin stimulation.

APCs were purified by removing CD4⁺ (clone GK1.5) and CD8⁺ T cells (clone T1B105) using mAb hybridoma supernatants, followed by magnetic bead (conjugated with goat anti-Rat IgG) separation. CD4⁺ T cells were purified by removing CD8⁺ T cells (mAb T1B105), MHC class II⁺ cells (mAb 10.2.16) and B cells (anti-mouse IgM and IgG (Qiagen)) using mAb hybridoma supernatants and magnetic bead separation. CD8 T cells were purified similarly, except that anti-CD4 (clone GK1.5) was used instead of anti-CD8 mAb. Individual B cells, CD11b⁺ cells and CD11c⁺ cells were purified by EasySep Mouse B cell enrichment kit, Mouse CD11b positive selection kit and Mouse CD11c positive selection kit II, respectively, from STEMCELL Technology according to manufacturer’s instructions. The purity of the cells was routinely ≥90%, analyzed by flow cytometry.