Spleens and Peyer’s patches (PP) were removed and passed through a cell strainer (90 µm, Falcon) to obtain a single cell suspension. Splenocytes were treated with red blood cell lysing buffer (Sigma-Aldrich), washed and resuspended in complete medium: RPMI 1640 with FCS (10%, Gibco) and penicillin–streptomycin (100 U/ml and 100 µg/ml, respectively, HyClone). Cells from PP were cultured in complete medium but with additional gentamicin (50 µg/ml, Gibco) to prevent bacterial contamination. After isolation, cells from spleen and PP (3–5 × 106 cells/ml) were either left unstimulated or polyclonally stimulated with αCD3 (1 µg/ml, Biolegend) and αCD28 (5 µg/ml, BioLegend) for 24 h. Cell culture supernatants were harvested and stored at −80°C.
Colonic lamina propria (cLP) leukocytes were isolated as previously described (17 (link)). In brief, colon tissue was incubated with EDTA (5 mM) to eliminate epithelial cells, followed by digestion with collagenase 8 (0.5 mg/ml, Sigma Aldrich) and DNase 1 (40 µg/ml, Roche). The released cells were layered on a 30/40/70% Percoll gradient (GE Healthcare).
Colonic lamina propria (cLP) leukocytes were isolated as previously described (17 (link)). In brief, colon tissue was incubated with EDTA (5 mM) to eliminate epithelial cells, followed by digestion with collagenase 8 (0.5 mg/ml, Sigma Aldrich) and DNase 1 (40 µg/ml, Roche). The released cells were layered on a 30/40/70% Percoll gradient (GE Healthcare).
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