Horizon fixable viability stain 450

Multi-colour flow cytometry analysis was performed as described previously [19 (link)]. Briefly, cells were incubated with a cocktail of monoclonal antibodies against rabbit CD4 (IgG2a, KEN-4), CD8 (IgG1, 12C.7) and IgM (IgG1, NRBM) on ice for 10 min. Cells were washed and further incubated for 10 min on ice with isotype-specific phycoerythrin (PE)-conjugated rat anti-mouse IgG1 (A85-1, BD) and biotinylated rat anti-mouse IgG2a (R19-15, BD) antibodies. After a third wash, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit T cells (KEN-5), washed with allophycocyanin (APC)-conjugated streptavidin (BD), and suspended in 7-AAD. Antibodies were from AbD-Serotec. Bovine CD8⁺ T cells were identified as previously described [18 (link)], and dead cells detected with BD Horizon Fixable Viability Stain 450 (BD biosciences). Data were acquired using a Fortessa X20 flow cytometer (BD) and analyzed using Flowjo v10.0.7 (Treestar).

Cell lines were maintained in RPMI1640 media supplemented 10% fetal bovine serum (FBS) (Atlanta Biologics, Cat. #S11150) and antibiotic-antimycotic solution (100X, GIBCO, Grand Island, NY, USA) at 37°C, 5% CO₂. JLAT and JLAT-TLR2 cells were plated at 100,000 cells/well in 96-well U-bottom culture plates and incubated with the following conditions for 16 hours: PBS, PMA (200 ng/mL), PIM6 (10 μg/mL), H37Rv lysate (100 μg/mL), LAM (100 μg/mL), H37Ra (MOI 35:1) and M. smegmatis (MOI 35:1). For assays involving inhibitors, cells were pre-incubated for 30 minutes at 37°C in media containing PAb-hTLR2 antibody (20 μg/mL; InvivoGen, Cat. Code pab-hstlr2) or BAY 11–7082 (0.30 μM; EMD Millipore Calbiochem™, Cat. #19-687-010MG). Cells were stained for viability (BD Horizon™ Fixable Viability Stain 450) and fixed with 1% paraformaldehyde. Viability and GFP expression was measured using a BD FACS Canto.

Cell suspensions were incubated with BD Horizon™ Fixable Viability Stain 450 (BD Biosciences, San Jose, CA, USA; Cat # 562247) at 4 °C for 30 min in the dark except the intestinal tissue and washed with ice-cold PBS. For the intestine, Fixable Viability Stain 520 (BD Biosciences; Cat # 564407) was used at 4 °C for 15 min in the dark. After adding anti-mouse CD16/CD32 (Thermo Fisher Scientific; Cat # 14-0161-85) to the samples, they were kept for 20 min in the fridge followed by a washing step. Subsequently, cells were stained with fluorochrome-conjugated anti-mouse antibodies at 4 °C for 30 min in the dark. Afterwards, FACS Flow^TM (BD Biosciences; Cat # 342003) was added for washing.

THP-1 cells, a monocytic cell line, were obtained from ATCC (Cat#TIB-202). HyClone™ RPMI 1640, kanamycin sulfate, Corning™ Accutase™ detachment solution and phorbol 12-myristate 13-acetate (PMA) were obtained from Fisher Scientific (cat. # SH30011.03, BP906-5, MT25058CI, and BP685-1, respectively). Fetal bovine serum was purchased from Atlanta Biologicals (cat. # S11150). BD Horizon™ Fixable Viability Stain 450 was obtained from BD Biosciences (cat. # 562,241).

RNA probes to detect OROV were designed to align at segment L genome and antigenome. First, Vero CCL81 cells were infected with OROV with both MOI 1 and MOI 10. RNA detection by RNA PrimeFlow™ assay was performed as indicated by the manufacturer. Briefly, infected cells were washed twice with FBS 2% in PBS 1×, fixed and permeabilized with PrimeFlow reagents. Then, RNA probes for genome (VF1-6000635) and antigenome (VF4-6000634) were added for hybridization at 40 °C. After that, three steps of signal amplification were performed: preamplification, amplification and label probes hybridization. Infection kinetics in Vero cells were also evaluated by qRT-PCR and FFA. Infected THP-1, Jurkat and Jeko-1 cells were also submitted to RNA PrimeFlow™ protocol described above. And finally, human PBMC infected in vitro were submitted to RNA PrimeFlow™ protocol, with some modifications. First, cells were stained for viability using BD Horizon™ Fixable Viability Stain 450, according to the manufacturer’s protocol. After that, cells were washed with FBS 2% in PBS 1× and incubated at 4 °C with antibodies for surface markers anti-CD14 PE, anti-HLA-DR APC-H7, anti-CD3 V500 and anti-CD19 PE CF594. Finally, cells were submitted to the RNA PrimeFlow™ protocol. Samples were analyzed in a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA).

PBMCs from healthy donors were cultured overnight in RPMI-1640 (Gibco, Waltham, MA, USA) supplemented with 10% FBS and 1% Penicillin/Streptomycin (GE Healthcare, Chicago, IL, USA) and stimulated with 35 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, St. Louis, CA, USA) and 1 µg/mL of ionomycin (Merck Millipore, Burlington, VT, USA) at 37 °C, 5% CO₂. Cells were then stained with BD Horizon™ Fixable Viability Stain 450 (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min in ice, followed by staining with the antibodies anti-CD45-PercP, anti-CD8-PE, and anti-HLA-DR-APC. Cells were sorted into two populations: CD45+/CD8+/HLA-DR+ and CD45+/CD8+/HLA-DRnegative in a FACS Aria III (BD Biosciences, Franklin Lakes, NJ, USA) with an efficiency above 90%.

Adherent THP-1 cells were incubated with Accutase™ for 15 minutes at 37°C. THP-1 cells were transferred to 5-mL tubes and washed with PBS. Cells were resuspended in BD Horizon™ Fixable Viability Stain 450 (0.25 μg/mL) and incubated at 4°C for 30 minutes. Cells were then fixed in 2% formaldehyde for 30 minutes at 4°C. Cells were analyzed using a FACS Canto. Percent infection by DHIV was quantified as a subset of the live population (FSC/V450/50-). Gates for infection were set according to the uninfected “mock” THP-1 cell controls. Three independent biological replicates were completed for all treatment conditions, each in triplicate wells per experiment. Population analysis was then done using FlowJoTM v10.7, to assess if infection levels and cell viability were consistent similar in all replicates. The Flow Cytometry figures are representative plots obtained from one of the replicates.