40 μm pore cell strainer

A single-cell suspension was prepared by filtering the homogenate using a 40-μm pore cell strainer (BD Pharmingen, San Jose, CA, USA). Cells were incubated with Fc Block anti-CD16/32 (IC1918F, BD Pharmingen) and stained with fluorescein isothiocyanate-conjugated anti-fibronectin or isotype control (IC002F, R&D Systems) for 1 h. Fibronectin-positive cells and E-cadherin-positive cells were analyzed using a BD FACS Diva instrument (version 8.0; BD Biosciences). Fibrosis was induced in hTECs with 2 ng/mL rTGF-β (R&D Systems) for 48 h. To evaluate the role of CCL8 in fibrosis, rTGF-β-stimulated hTECs were simultaneously treated with an anti-CCL8 monoclonal antibody (50 or 100 ng/mL; Invitrogen). To quantify cell fibrosis and adhesion, cells were harvested and stained with antibodies against fibronectin (Invitrogen) and E-cadherin (R&D Systems) according to the manufacturers’ protocols. Apoptosis and necrosis were quantified by flow cytometry using an annexin V/propidium iodide assay. hTECs stained with propidium iodide and FITC-conjugated annexin V were incubated for 30 min in the dark, followed by analysis with the BD FACS Diva instrument.

Isolated crypts were incubated in culture medium for 45min at 37°C, followed by cell dissociation using Accutase (STEMCELL) for 15min at 37°C. Dissociated cells were filtered through a 40μm pore cell strainer (BD Bioscience). TdTomato ^positive cells were sorted by flow cytometry using an iCyt Synergy Flow sorter (Sony Biotechnology). Single viable epithelial cells from both the intestine and colon were gated by forward scatter, side scatter, pulse-width parameter and by negative staining for DAPI (Sigma Aldrich). Sorted cells were collected in crypt culture medium, embedded in Matrigel and then overlaid with complete culture media.

The lungs of the mice were dissected out at 1, 2, and 3 dpi, and cell suspensions were prepared by mechanical dissociation in 3 mL of cold (4°C) staining buffer (2.5% Fetal Calf’s Serum in PBS). No enzymatic digestion by collagenase treatment was used, since preliminary experiments showed an important reduction in the recovery of myeloid-gated cells and CD11b+ cells after such treatment (Supplementary Material). The lung tissue was cut into small pieces and cell suspensions were prepared by mechanical dissociation, disrupting the tissue and filtering through a 40 μm pore cell strainer (BD Biosciences). The filtrate was then centrifuged for 5 min at 110 g and 4°C in order to obtain the lung cells. The material recovered was analyzed by flow cytometry (see below), and the S. pneumoniae CFUs and cytokines in the supernatants were quantified. Colony-forming units were determined by growing serial dilutions of the lung supernatant on Mueller-Hinton sheep blood agar plates (BD Biosciences, San Jose, CA), counting the colonies formed after a 24 h incubation at 37°C in an atmosphere of 5% CO₂. Dissemination of the bacteria to other organs was determined by CFU quantification in samples from spleen and olfactory bulbs at 3 dpi (50 ± 7 CFUs, n = 6 in splenic samples of infected BL10).