Facs lsrfortessa cytometer

Mononuclear cells were stained with various combinations of the following fluorescence-conjugated antibodies: CD19, CD1d, CD5, CD21, CD23, CD4, CD8, CD25, CD95, GL-7, CD138, PD-1, CXCR5 and B220 (BD Biosciences, San Diego, CA, USA). These cells were also intracellularly stained with the following antibodies: IFN-r, IL-4, IL-2, IL-10, IL-17 and Foxp3 (all eBioscience, San Diego, CA, USA). Before intracellular staining, the cells were restimulated for 4 hr with 25 ng/mL PMA (Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences). Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed using a FACSCalibur and FACS LSRFortessa cytometer (BD Biosciences).

Tumors were dissociated using a mouse tumor dissociation kit with the gentleMACS Octo Dissociator according to the manufacture’s protocol (Miltenyi Biotec). Draining lymph node (dLN) cells and spleen cells were dissociated in RPMI1640 supplemented with 5% FBS. Tumor infiltrating immune cells, dLN cells and spleen cells were prepared as single cell suspensions and immunostained with various combinations of the following fluorescence-conjugated Abs: CD3, CD4, CD8, DX5, F4/80, CD11c, CD206, CD40, CD80, PD-L1, IFN- γ, and granzyme B. Intracellular markers were identified using Abs for IFN-γ and granzyme B (eBioscience, San Diego, CA, USA). Prior to intracellular cytokine staining, cells were incubated in culture medium containing PMA (25 ng/ml; Sigma-Aldrich, St. Louis, MO, USA), ionomycin (250 ng/ml; Sigma-Aldrich), and monensin (GolgiStop, 1 μl/ml; BD Biosciences, Franklin Lakes, NJ, USA) in a 5% CO₂ atmosphere at 37 °C for 4 h. Intracellular staining utilized an intracellular staining kit (eBioscience) as per the manufacturer’s guidelines. Analysis was conducted using a FACS LSR Fortessa cytometer (BD Biosciences) and FlowJo software (BD Biosciences).

Spleens were removed aseptically and transferred to Hanks Balanced Salt Solution (HBSS). Single-cell suspensions were obtained by pressing the spleens through a 70-µm nylon cell strainer, followed by centrifugation and two washes in HBSS. Approximately 2 × 10⁶ splenocytes were transferred to U-bottom 96-well microtiter plates and were incubated for 20 min (4°C in the dark) with PE-conjugated H-2D^b tetramers for ZIKV E_294–302 (25 (link)) and subsequently stained for an additional 20 min (4°C in the dark) for relevant cell-surface markers. Cells to be stained for intracellular granzyme B were then permeabilized using the BD Biosciences FoxP3 staining protocol. Next, the cells were centrifuged, washed, fixed in 1% PFA, and finally resuspended in PBS and stored at 4°C until flow cytometric analysis. Cell samples were analyzed using FACS LSRFortessa cytometer (BD Biosciences), and the data were analyzed using FlowJo software version 10 (Tree Star).

PD-L1+ or PD-L1- MDSCs were isolated from MRL/MpJ mice. CD4+T cells were isolated from spleen of MRL/lpr mice by using the CD4+ T cell microbeads. Isolated PD-L1+ or PD-L1- MDSCs were cocultured with Cell Trace-violet -labelled CD4+ T cells (MDSC:T cell ratio 1:1, 1/2:1, 1/5:1). CD4+T cells were stimulated by 1 μg/ml of anti-CD3/CD28 (eBioscience, San Diego, CA, USA) and 30U/ml of recombinant IL-2 (R&D Systems, Minneapolis, MN, USA). After 5 days, proliferation of T cells was analyzed using FACS LSRFortessa cytometer (BD Pharmingen, San Jose, CA, USA).

Approximately 1–5 × 10⁶ brain/spleen/blood cells were transferred to U-bottom 96-well microtiter plates and were incubated for 20 min (4°C in the dark) with PE-conjugated H-2D^b tetramers for ZIKV E_294–302 (34 (link)) and subsequently stained for an additional 20 min (4°C in the dark) for relevant cell-surface markers. Next, the cells were centrifuged, washed, fixed in 1% PFA and finally resuspended in PBS and stored at 4°C until flow cytometric analysis. Cell samples were analyzed using FACS LSRFortessa cytometer (BD Biosciences), and the data was analyzed using FlowJo software version 10 (Tree Star).