Fluorescein isothiocyanate (fitc)

Flow cytometric characterisation of control and treated ucMSC was performed by labelling with standard MSC markers, according to ISSCT criteria [41 (link)]: CD13 (PE-Cy7, BD Biosciences, San Jose, CA, USA), CD73 (PE, BD Pharmingen, San Diego, CA, USA), CD90 (APC, R&D Systems) and CD105 (FITC, R&D Systems), hematopoietic markers CD31 (PB, BD Biosciences), CD45 (APC-Cy, BD Biosciences) and the surface markers: HLA class I (Pacific Blue, Biolegend, San Diego, CA, USA), HLA class II (PERCP, BD Biosciences), CD362 (APC, R&D Systems), PD-L1 (PE, Biolegend), CD271 (PE-Cy7, BD Pharmingen), CD73 (PE, BD Pharmingen) and CD40 (PERCP, Biolegend).

Hep3B cells were trypsinised (0.05% trypsin/0.53 mM EDTA), washed, and adjusted to 1 × 10⁶ cells/ml (in saline containing 1% albumin; Biological Industries, Israel) for 10 min to determine their purity. The cells were fixed with 4% paraformaldehyde and permeabilised in 0.1% saponin in phosphate-buffered saline (PBS) for 20 min at room temperature. They were then stained with an anti-human HBsAg monoclonal antibody (R&D System) for 30 min at room temperature. The cells were then incubated with propidium iodide (PI) to stain fragmented DNA and Annexin V conjugated to fluorescein isothiocyanate (FITC) (R&D systems) to stain phosphatidylserine (PS) according to the manufacturer's instructions. The cells were analysed by flow cytometry (Becton-Dickinson LSR 11, Immunofluorometry Systems, Mountain View, CA, USA). Apoptotic cells were defined as Annexin V (+)/PI (-). Viable cells were defined as Annexin V (-)/PI (-). In each experimental setting, unstained controls, immunoglobulin G (IgG) isotype controls, and FMO controls were used [41 (link)].
To analyse the cell cycle, Hep3B was fixed in cold 70% ethanol for at least 30 min at 4°C. After washing two time in PBS, the cells were treated with 50 μl RNase (100 μg/ml), stained with 5 μl propidium iodide (PI) (50 μg PI/100 ml solution), and analysed with flow cytometry [41 (link)].

For isolation of peritoneal macrophages, mice were injected with 5 ml PBS. Macrophages were isolated from peritoneal lavage by plastic adherence. For isolation of granulocytes, mice were injected intraperitoneally with 1.5 ml sterile thioglycollate (3%). Elicited cells were harvested 4 h later by peritoneal lavage with 5 ml of cold PBS, and neutrophils were further purified from peritoneal lavage by magnetic cell sorting (Miltenyi Biotec). FITC-labeled E. coli were prepared by incubation with 0.5 mg/ml FITC (Sigma) for 20 min at 37 °C. Peritoneal macrophages (1 × 10⁵ cells) or neutrophils (1 × 10⁶ cells) were incubated with FITC-labeled bacteria at a multiplicity of infection of 100 for 30 min at 37 °C. After washing steps, cell nuclei were stained with DAPI (Invitrogen), followed by visualization using confocal laser scanning microscopy (LSM 510, Zeiss). The ratio of engulfed bacteria (as determined by overlay of green bacteria) was quantified by an independent researcher from 300 counted cells per well. In some experiments, peritoneal macrophages and neutrophils were pretreated with recombinant human IL-26 (100 ng/ml, R&D systems) before infection by FITC-labeled E. coli.