Live dead fixable aqua dead cell stain kit

After a 4-hour cytotoxicity assay, cells were washed with PBS (Gibco) and stained for dead cells using Live/Dead^® Fixable Aqua Dead Cell Stain Kit (Molecular Probes™) for 30 minutes on ice in the dark. Cells were further washed with PBS buffer (PBS, 1% FCS) and fixed with 1% paraformaldehyde in PBS solution. For NK cell phenotyping, expanded NK cells were harvested after culturing in either 500, 2000, or 4000 mg/L glucose for 4 days. Subsequently, NK cells were washed in PBS and first stained for dead cells using Live/Dead^® Fixable Aqua Dead Cell Stain Kit (Molecular Probes™) for 30 minutes on ice in the dark, and subsequently stained for the following surface markers for 30 minutes on ice and in the dark: CD3-APC-Vio770, -VioBlue or -PerCP-Vio700; CD56-PE-Vio770 or -APC-Vio770; KIR2DL2/3-PE; KIR3DL1-PerCP; NKG2A-APC; KIR2DL1-FITC; NKp30-PE-Vio770; NKp44-VioBright; NKp46-PE; NKG2D-APC; PD1-VioBright; DNAM1-PE; CD96-PE-Vio770; NKG2C-APC; LAG3-VioBlue; TIM3-APC; TIGIT-PE. FMO controls were stained for Live/Dead, CD3 and CD56. All antibodies were purchased from Miltenyi Biotech. All flow cytometric analyses were performed with BD FACS Canto II. Data were analyzed with FlowJo 10.1r5 64-bit software.

To isolate pDCs, cDCs, and monocytes, human PBMCs were labeled with Alexa 647 anti-human CD11c (301620, clone 3.9, BioLegend), anti-human HLA-DR (Class III) PE-Texas conjugate (MHLDR17, Life Technology), BV421 anti-human CD14 (325627, clone HCD14, BioLegend), PE-Cy7 anti-human CD123 (560826, clone 7G3, BD Biosciences), APC-Fire 750 anti-human CD8a (301065, clone RPA-T8, BioLegend), APC-Fire 750 anti-human CD20 (302357, clone 2H7, BioLegend), APC-H7 anti-human CD3 (560275, clone SK7, BD Pharmingen) and Live/Dead Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen). Labeled cells were fixed with 4% PFA in PBS for 5 min, then cell marker expression was analyzed and cells were isolated with BD FACSAria II (BD Biosciences). The gating strategy is shown in Supplementary Figure S4.
To isolate CD4⁺ T, CD8⁺ T, NK, and B cells, human PBMCs were labeled with APC-H7 anti-human CD3 (560275, BD Bioscience), PE-Cy7 anti-human CD4 (317413, Biolegend), PE anti-human CD56 (355503, Biolegend), FITC anti-human CD8 (300905, Biolegend), APC anti-human CD19 (302211, Biolegend), and Live/Dead Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen). Labeled cells were fixed with 4% PFA in PBS for 5 min, then expression markers were analyzed and cells were isolated with BD FACSAria II (BD Biosciences). The gating strategy is shown in Supplementary Figure S4.

Washed cells were stained with aqua LIVE⁄DEAD® Fixable Dead Cell Stain Kit, (Invitrogen), CD3-Pe-Cy7 (e-bioscience), CD11b-APC-Cy7 (BD bioscience) and FITC anti-human CX3CR1 (Cambridge Bioscience) at 1:100 dilutions for 30 min. Unstained controls were used to set voltages, single-stained controls for compensation and fluorescence minus one (FMO) controls for gate setting during analysis. The samples were run on a Becton Dickinson LSR Fortessa and analysed using FlowJo 9.3 (^©TreeStar,Inc). Events were gated on live singlets then analysed for cell surface marker expression. Cells infected with Mtb H37Rv-FITC were stained with aqua LIVE⁄DEAD^® Fixable Dead Cell Stain Kit (Invitrogen) and then acquired on Becton Dickinson LSR Fortessa before analysis with FlowJo 9.3. The murine lung flow cytometry panel was composed of annexin-V/propidium iodide (AbCam), CD11b-APC-Cy7.7 (Becton Dickinson) and CD3-Pacblue (Cambridge Bioscience). For the single stained controls anti-rat/hamster murine beads were used (Invitrogen) and an unstained and FMO controls were also carried out. The samples were run on a Beckman Coulter CyAn ADP and live singlet cells were gated and analysed using Flowjo 9.3.

Spleen, popliteal lymph node (pLN) and joint footpad of CHIKV-infected mice were harvested at 6 dpi and 7 dpi. Isolation of splenocytes, pLN and joint footpad cells were processed as described4 (link)21 (link)26 (link). Isolated cells from spleen, joint footpad and pLN were first stained with Live/dead Fixable Aqua Dead Cell Stain Kit (1:400) (Life Technologies) for 20 min. Cell were then washed and resuspended in staining buffer (2% fetal bovine serum in PBS). Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience). Intracellular staining of rat anti-Foxp3 (FJK-16s, eBioscience) was done according to manufacturer’s instructions. Data acquisition and analyzes were done as described above.

Endothelial cells (ECs)—HUVEC or HDMECs—were plated on 6-well plates. Six hours later, stably mCherry-expressing A673 human sarcoma cells (A673-mCherry) were collected and infected with 0.1 MOI rHSVQ or RAMBO virus for 30 min in a suspension condition. Cells were washed and overlaid on top of an equivalent number of ECs and cultured for 24 h. Cells were then collected and stained with Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies, Eugene, OR, USA) and analyzed in CytoFlex (Beckman Coulter, Brea, CA, USA). Single stain controls for each fluorochrome were prepared using cells or compensation beads (Invitrogen, Waltham, MA, USA) for compensation. Tumor and endothelial cells were initially separated by gating for mCherry positive population (550–650 nm of emission), and then further gated for GFP expression and Live/Dead cell staining-positive polulation. GFP expression represents virus infected cells while Live/Dead staining represents dead cell population. Data were analyzed using FlowJo v10.7 (FlowJo LLC, Ashland, OR, USA).