Macsquant16 cytometer

We evaluated the cell viability and phenotype in the different stages of the procedure by flow cytometry. Briefly, cell surface markers staining was followed by staining with Fixable Viability Dye-eFluor450 (eBioscience). Then, the cells were fixed and permeabilized using the FOXP3 transcription factor staining kit (eBioscience) for intracellular staining. All the antibodies are listed in Supplementary Table 2. Flow cytometry analysis of labeled cells was performed with a MACSQuant16 cytometer (Miltenyi Biotec), acquiring at least 100,000 events, and the data were analyzed using Kaluza software (Beckman Coulter).
To isolate the CD4⁺ single-positive (SP) and CD4⁺CD8⁺ double-positive (DP) thyTreg cells, 50x10⁶ of total thyTreg cells were labeled with anti-CD4-VioBlue (Miltenyi Biotec) and anti-CD8-FITC (Beckman Coulter). Cells were washed and resuspended at 5x10⁶ cells/ml in MACSQuant Tyto Running Buffer (Miltenyi Biotec) and were subjected to two consecutive rounds of sorting with High-Speed MACSQuant Tyto Cartridges (MACSQuant Tyto cell sorter, Miltenyi Biotec). After the first round, CD4⁺SP cells were collected from the positive fraction. The negative fraction was loaded into a second cartridge, and CD4⁺CD8⁺ DP cells were collected from the positive fraction.

For flow cytometric analysis, human monocyte-derived macrophages (M1 and M2) were blocked for 30 min at 4°C with 50% FBS in autoMACS Rinsing Solution supplemented with MACS BSA Stock Solution (Miltenyi, Germany) and incubated with fluorescently labeled antibodies directed against, CD68 (Clone: REA886, Miltenyi Germany), CD80 (Clone: REA661, Miltenyi Germany), CD209 (Clone: REA617, Miltenyi Germany), and monoclonal mouse IgG2B anti-human VISTA (Clone: # 730804, R&D Systems, USA) as well as corresponding isotype control antibodies for 30 min at 4°C. Dead cells were stained with Propidium Iodide Solution (Miltenyi Biotec, Germany). The cell suspension was analyzed using a MACS Quant 16 cytometer (Miltenyi Biotec, Germany). Data were evaluated using Flowlogic 7.3 software (Inivai™ Technologies, USA).

Human neutrophils were plated at a density of 8 × 10⁵ cells/ml. Fixed C. difficile was incubated with one mAb at 20 µg/mL or a cocktail of mAbs NF10, KH2, 1E2, 2B7, and TG10 at an equimolar ratio and stained with pHRodo dye (Thermo Fisher Scientific) following the manufacturer’s instructions. An irrelevant mouse IgG (in-house produced) was used as an isotype control. Bacteria were then incubated with neutrophils at a Multiplicity Of Infection (MOI) of 100 for 1.5 h at 37°C (20,000 neutrophils for each condition). Flow cytometry acquisition was performed on a MacsQuant16 cytometer (Miltenyi, Germany) and analyzed using FlowJo software v10.8.1 (BD Biosciences, CA, USA).