Cd19 6d5

Colons were dissected, washed with ice-cold PBS and cut into small pieces. Colons pieces were incubated with PBS containing 1 mM DTT, 5 mM EDTA and 10 mM HEPES at 37°C for 30 min with gentle shaking to remove the epithelial layer. The colon segments were further digested in RPMI medium containing 0.5 mg/ml collagenase D at 37°C for 1.5 hr. The supernatant was passed through 70 µm cell strainer and enriched by 37.5% Percoll to isolate lamina propria cells.
The following monoclonal antibodies were used for flow cytometry: CD4 (RM4–5; 14–0042–85), CD11b (M1/70; 48–0112-82) and CD8a (53-6.7; 48–0081–82) from Affymetrix eBioscience, CD19 (6D5; 115512), NK1.1 (PK136; 108708), CD11c (N418; 117306), Gr1 (RB6–8C5; 108426) and F4/80 (BM8; 123109) from BioLegend. The dilution factor for all antibodies was 1:300. The following gating strategies were used: B cells were gated as live cells and CD19⁺. CD4⁺ T cells were gated as live cells, CD4⁺ and CD8⁻. CD8⁺ T cells were gated as live cells, CD8⁺ and CD4⁻. NK cells were gated as live cells and NK1.1⁺. Macrophages were gated as live cells, CD11b⁺, Gr1^low-neg, F4/80⁺ and CD11c⁻. Neutrophils were gated as live cells, CD11b⁺ and Gr1^hi. CD11b⁺CD11c⁺ cells were gated as live cells, CD11b⁺, Gr1^low-neg, CD11c⁺ and F4/80⁻. Flow cytometry data were acquired on a BD FACSCalibur and analysed using TreeStar FlowJo software.