Fortessa x20 cytometer

Cells were washed in FACS buffer (0.05% BSA, 2 mM EDTA in PBS, pH 7.4) blocked using anti-CD16/32 (1 µg; Biolegend) for 15 mins at 4°C, followed by 30 min antibody staining for the surface markers with isotype controls. Cells were again washed and resuspended in 200 µL of FACS buffer without fixation. Antibodies used are listed in Table 1, all bought from BioLegend apart from FITC-CD8a, which was bought from BD Biosciences.
Intracellular staining was performed as per manufacturer’s instructions using eBioscience FOXP3/Transcription Factor Staining Buffer Set (invitrogen). Briefly, after the last wash of the extracellular staining protocol, cells were fixed in Fixation/Permeabilization Buffer for 30 min at room temperature. Cells were washed and resuspended in Permeabilization Buffer; intracellular antibodies were added for 30 min in room temperature. Cells were again washed in Permeabilization Buffer and resuspended in FACS buffer for flow cytometric analysis.
Data were acquired using BD Fortessa X20 cytometer and Diva software (BD Biosciences) and then analysed using FlowJo (Tree Star Inc, USA) software. Fluorescence minus One (FMO) controls were used for gating and single stains for compensation were obtained using UltraComp eBeads™ Compensation Beads (InVitrogen) for liver, spleen and blood panels and single stained cell samples for peritoneal cells.

Cells were washed in PBS then stained with LIVE/DEAD™ fixable violet dead cell stain (ThermoFisher Scientific). Next, cells were washed with FACS buffer (PBS, 0.5% BSA, 2 mM EDTA), Fc receptors were blocked with TruStain FcX (Biolegend), and then cells were incubated with antibody cocktail prepared in a 1:1 mix of FACS buffer and BD Horizon™ Brilliant Stain Buffer. Cells were washed in FACS buffer, fixed in BD fixation/permeabilization solution, washed in FACS buffer and stored at 4°C until analysis. Equal numbers of Precision Count Beads™ (Biolegend) were added to samples just before analysis to determine total cell numbers per organ. A BD Fortessa X-20 cytometer was used for all experiments. Flow cytometry data were collected with FACSDivaTM software (BD) and FCS files were analyzed using FlowJo V10 (BD). Antibody probes from BD included (target, clone, fluorochrome): B220, RA3-6B2, BUV737; CD3ϵ, 145-2C11, PE; CD8α, 53-6.7, BV711; CD11c, HL3, BUV395; CD45, 30-F11, FITC; Siglec-F, E50-2440, BV786. Antibody probes from Biolegend included (target, clone, fluorochrome): CD4, GK1.5, BV510; CD11b, M1/70, PE-Cy7; CD19, 6D5, PE-Dazzle594; Ly6C, HK1.4, APC-Cy7; Ly6G, 1A8, PerCP-Cy5.5; MHC-II (I-A^b), AF6-120.1, APC.

One million C1498-FLuc cells in vitro were treated with vehicle (DMSO) or 5-Aza (5 μM) for 48 h. One hundred thousand live cells were counted, washed, and stained with anti-Annexin V antibody (Alexa Fluor 488) and propidium iodide (kit V13241) (Thermo Fisher Scientific; Waltham, MA, USA) and subjected to flow cytometric analysis on the BD Fortessa X20 cytometer. Data were analyzed using FlowJo Version 10 (Becton, Dickinson & Co.; Franklin Lakes, NJ, USA).

Cells were transiently transfected as described above. 18 hrs following transfection cells were washed with PBS and arrested in S-phase with double thymidine block. After the blocking fresh medium with 10% FBS 1% P/S was added and cells were incubated at 37°C in 5% CO₂ for 1–8 hrs., for starvation conditions media was changed to 0.5% serum after a 1 hr release. Cells were collected and stained with BD Transcription Factor Buffer Set for Intracellular staining (cat # 562574), using Flag primary and Alexa 488 secondary antibodies. The DNA was labelled with propidium iodide (PI). Cells were analyzed using BD Fortessa X20 cytometer. The single cell population was gated for flag expression and the flag positive (Tuberin expressing) population in G2/M phase was visualized for cell size using forward scatter (FSC-A).

Intracellular cytokine secretion by antigen-specific T cell was analyzed using multiparameter flow cytometry. Briefly, cryopreserved PBMCs were thawed, washed and rested overnight in R10 medium at 37°C in a humidified incubator. Cells were stimulated with HCV NSmut (1 μg/ml), or HIVconsv peptide pools (2 μg/ml), mock control (0.45% DMSO) and positive controls (SEB, 5 μg/ml; CMV pp65, 2 μg/ml, NIH AIDS Reagent Repository) at 37°C for 6 h in the presence of Golgiplug, Golgistop (BD Biosciences) and CD107a BV421. Following viability and surface staining, cells were then fixed using BD cytofix/cytoperm solution according to the manufacturer's instructions and intracellularly stained using reagents as listed in Supplementary Table 1. At least 10,000 viable singlet CD3⁺ CD4⁺ or CD8⁺ lymphocyte events were acquired using a BD Fortessa X20 cytometer. Data were analyzed using FlowJo v9.9.3 (FlowJo, US) and GraphPad Prism v7.0.

For in vivo peritoneal recruitment experiments mice underwent intraperitoneal injected with 4% thioglycolate. 4 days later mice were killed and the peritoneal cavity was lavaged with 5 mL of PBS containing 5 mM EDTA. Macrophages were plated into serological Petri dishes in DMEM/F12 (with 2% FCS, Pen/Strep and Glutamine) and allowed to adhere for 75min. Adherent macrophages were harvested by washing the plates briefly with 2 PBS washes prior to detachment with ice cold PBS/5 mM EDTA then plated for cell activation assays. In vivo macrophages were stained with antibodies against CD45 (FITC), CD11b (PerCP) and F4:80 (APC) to assess the purity of the population, specificity of the antibody staining was confirmed using isotype control antibodies stained with the same fluorochromes (all antibodies and isotype controls Biolegend, UK). All flow cytometry was performed using a BD Fortessa X20 cytometer and Diva software (BD Biosciences, Oxford, UK). Data was analyzed using Flow Jo software (TreeStar Inc, Wokingham, UK).

Measurement of protein levels of secreted IL‐1β, TNFα, and IL‐6 in cell supernatants from 1.5 × 10⁶ human MoDM's was performed by Legend Plex multi‐analyte assay kit (BioLegend) according to the manufacturer's instruction. Data were acquired using a BD Fortessa X20 cytometer (BD Biosciences) and analysed using LegendPlex Software (BioLegend).