Lsrfortessatm cytometer

Twenty-four hours after the end of treatment, blood samples were collected to quantify circulating cytokines levels. Titrations were performed using a bead-based immunoassay LEGENDplex^® multi-analyte flow assay kit (BioLegend, Paris, France), according to the manufacturerʼs protocol. Cytokine concentration was determined using standard curves obtained using recombinant cytokine standards provided in the kit. Samples were analyzed on a LSRFortessa^TM cytometer (BD Biosciences, Le-Pont-de-Claix, France).

Cytokine quantification of Th1/Th2/Th17 cytokines; IL-2, IL-4, IL-6, IL-10, TNF, IFNγ, and IL-17A was performed with the aid of the CBA Human Th1/Th2/Th17 cytokine kit (BD Biosciences, San Diego, CA United States) and using the supernatants of cells that had been stimulated with either OMVs or PHA for 24 h, following the manufacturer’s instructions. Samples were read by using the LSRFortessa^TM cytometer (BD Biosciences) with CBA template, a total of 2000 events were recorded, and the resulting data was analyzed with the FCAP Array^TM software v3.0 (BD Biosciences).

Fluorescence dye-conjugated antibodies for the flow cytometry analysis include PE-conjugated anti-Human CD47(B6H12) antibody (BD, 556046; 5 μl per sample), PE-conjugated anti-mouse CD11b antibody (Biolegend, 101207; 1 μl per sample), APC-conjugated anti-mouse F4/80 antibody (R&D, FAB5580A; 1 μl per sample). About 2–10 × 10⁵ cells were resuspended in 100 μl PBS containing 2% FBS and stained with the indicated fluorescence antibodies for 30 min on ice in dark and the flow cytometry were then performed on LSRFortessaTM cytometer (BD Biosciences) or CytoFLEX cytometer (Beckman). The data were analyzed by using the Flowjo software. To calculate the “Relative MFI”, we measured the mean of fluorescence intensity (MFI) by using flowjo software and then calculated the ratio of MFI of RAGA knockdown cells to MFI of shControl cells or the ratio of MFI of RAGA overexpression cells to MFI of pBabe cells. The ratios were indicated as “Relative MFI”.

The cytotoxic effects of the DV2 peptide were evaluated by measuring cell metabolic activity as well as cell viability. Cell proliferation was assessed using the reagent WST-1 (Roche) according to the manufacturer’s instructions, and the number of live cells was determined by flow cytometry. Briefly, Vero CCL-81 cell monolayers established in 96-well plates were incubated at 37 °C for 24 h with increasing concentrations of the peptide ranging from 0.75 to 24 μM. Equivalent volumes of DMSO were used as the control. The WST-1 reagent was added to the wells, which were then incubated for 1 h at 37 °C. The absorbance was measured using a microplate reader at 440 nm. For flow cytometry analysis, live/dead aqua fluorescent reactive dye (Invitrogen) was added to the wells after a washing step for incubation for 30 min. at room temperature. The cells were then acquired on a LSR FortessaTM cytometer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (version 10, Tree Star, San Carlo, CA, USA).

Peripheral blood mononuclear cells were stimulated with either a 1, 10, or 25 μg/mL suspension of OMVs from both strains or a 5 μg phytohemagglutinin (PHA) solution for 12, 24, and 48 h. Plates were incubated at 37°C under 5% CO₂. Monoclonal antibodies (mAbs) coupled to the following fluorochromes were used: anti-human CD91-eFluor 660 (eBioscience), anti-human CD3-APC (BD Pharmingen^TM), anti-human CD19-PE-Cy7 (BD Biosciences), anti-human PD-L1-PE (BioLegend), anti-human PD-1-FITC (BioLegend), anti-human CD86-PE (BioLegend), anti-human CD69-TRI-COLOR (Molecular Probes). After stimulation, 100 μL of supernatant were collected from wells and stored at −70°C until usage. Then, PBMCs were collected and washed with FACS buffer, followed by incubation with diluted mAbs in FACS buffer for 1 h at 4°C (protecting the reaction from light). Finally, cells were washed with FACS buffer, fixed with PBS-PFA (Paraformaldehyde) 1%, and resuspended in 400 μL of FACS buffer. Samples were analyzed in LSRFortessa^TM cytometer (BD Biosciences), providing a total number of 30,000 events, and the resulting data was analyzed with the aid of the FlowJo^® software.

The cell monolayers were washed twice with PBS and trypsinized using 1× Trypsin/EDTA (Gibco). Cells were fixed/permeabilized using a Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer’s instructions, labeled with the 4G2 monoclonal antibody (Millipore) (10 µg/mL), and incubated with a rabbit anti-mouse IgG antibody coupled to AF488 (Thermo Fisher Scientific) (final dilution of 1:1000). Flow cytometry analyses were performed using an LSR FortessaTM cytometer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (version 10, Tree Star, San Carlo, CA, USA) to determine the percentage of infected cells.