Facsdiva 9

Cell-bound markers were evaluated at baseline and week 24 using flow cytometry. Briefly, cryopreserved PBMCs were thawed in RPMI 20% FCS, washed using PBS, and incubated with mouse monoclonal antibodies against CD4 (fluorophore BV510; clone RPA-t4; Biolegend, San Diego, CA, USA), CD8 (fluorophore PercP-CY5.5; clone SK-1; BD Pharmingen, Franklin Lakes, NJ, USA), CD38 (fluorophore APC; clone HIT-2; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), and HLA-DR (fluorophore BV711; clone G46-6; Biolegend, San Diego, CA, USA). Fluorescence minus one (FMO) controls were used to define positive gates for the expression of different proteins. The analysis was performed using BD FACS Diva 9.0.1. For soluble markers, a multiplex immunoassay was used, as previously described [27 (link),28 (link)]. In short, aspecific heterophilic immunoglobulins (IL-6, IL-1b, IP-10, MCP-1 MIP-1a, MIP-1b, sICAM-1, sCD14, sCD163, MIG) were pre-absorbed with HeteroBlock (Omega Chemicals, Hebron, IN, USA). Measurements were performed using a Bio-Rad FlexMAP3D in combination with xPONENT software version 4.1 (Luminex, Austin, TA, USA). Data analysis was performed using Bioplex Manager 6.1.1 (BIO-RAD).

For apoptosis analysis, siRNA-mediated KD was performed for 96 h before both the attached and non-attached cells were collected and stained using the “Annexin V-FITC Detection Kit” (PromoKine, PromoCell GmbH, Heidelberg, Germany) according to the manufacturer’s instructions. The samples were analyzed with FACS (“LSRFortessaTM”, BD Biosciences, Franklin Lakes, NJ, USA) and data interpretation was carried out with “BD FACSDiva 9.0” software (BD Biosciences, Franklin Lakes, NJ, USA).

Splenocytes were labeled with APC-Cy7-anti-CD3 (557596), BV421-anti-Gr-1 (562709), FITC-anti-B220 (553088), and PE-Cy7-anti-TER-119 (557915) antibodies (BD Biosciences, San Jose, CA, USA) in 2% normal rat serum and 1 μg/ml 2.4G2 anti-CD16/CD32 (BD Biosciences, 553142). Dead cells that stained with 7-AAD (BD Biosciences), plus doublets, identified by their FSC-A versus FSC-W profiles, were excluded from analyses as described [50 (link)]. Data were acquired using a BD FACSCantoII (BD Biosciences) cytometer and BD FACSDiva 9.0. Data were analyzed with FlowJo 10.7.1.

The flow cytometry was performed according to the procedures reported previously [52 (link)]. Briefly, an amount of 1 × 10⁶ cells/mL of EC109 was firstly infected by a volume of C. albicans SC5314 and C. glabrata ATCC15126 dual co-cultures (both at 1 × 10³ cells/mL) in the presence of 8 μg/mL SH and/or 0.125 μg/mL FLU at 37°C for 4 h. Secondly, the cells were incubated with Hypoxyprobe^TM at 37°C 5% CO₂ for 2 h and FITC-labeled monoclonal antibody-1 (1:100 dilution) at 37°C for 2 h. After twice washing with sterile PBS, the labeled cells were loaded in a BD FACSCelesta^TM flow cytometer (Franklin Lake, NJ, USA). The data were processed using BD FACSDiva 9.0 software at an excitation of 488 nm and an emission of 525 nm. A quantity of approximately 10,000 cells were acquired for the analysis.

The lt-NES cell cultures were harvested and washed before antibody incubation. Anti-O4 APC-conjugated antibody (Miltenyi, no. 130-119-155) was diluted 1:200 in FACS buffer (PBS + 2% fetal bovine serum + 2 nM ethylenediaminetetraacetic acid). Cells were incubated for 30 min at +4°C in darkness, followed by wash and incubation with propidium iodide (Life Technologies), a viability marker, diluted 1:1,000 in FACS buffer at least 5 min before acquisition. Cells were analyzed in an LSR II flow cytometer (Becton Dickinson) and results were analyzed with BD FACSDiva 9.0 software (BD Biosciences) and FlowJo v.10.8 Software (BD Life Sciences). The gating strategy is shown in Figure S1C.