Anti cd14 pacific blue

From synovial tissue samples, 5 × 10⁵ cells were stained with multitest CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC reagent (BD, PN 342417) and anti-CD14- PacificBlue (Invitrogen, PN MHCD1428) for 10 min at RT in the dark. The cells were washed with 3 ml PBS-E and finally resuspended in 200 μl PBS-E. The samples were either measured the same day or resuspended in 200 μl fixation buffer (3 ml PBS-E + 37,5 μl formaldehyde). The samples were analysed using a FACSVerse instrument (BD).

PBMC were prepared as described above, re-suspended in R10 medium at 10⁶ cells per ml and distributed into 5 ml FACs tubes (Invitrogen, UK) at 10⁶ cells per tube; one tube per stimulation condition. Stimuli were added to each tube as appropriate (R10 medium as a negative control; PPD at 20 μg/ml; SEB at 5 μg/ml; FEC peptides at 25 μg/ml) and samples were incubated at 37°C for 2 h. After this time, 3 μl of brefeldin A (BFA, Sigma, UK; stock concentration 1 mg/ml) was added to all tubes to give a final concentration of 3 μg/ml, and tubes were incubated for a further 18 h (overnight) at 37°C. Following stimulation, PBMC were washed in FACS buffer (PBS with 0.1% bovine serum albumin (Sigma) and 0.01% sodium azide (Sigma)) and stained with VIVID live/dead reagent (Molecular Probes) as well as with a surface stain cocktail of antibodies (anti-CD4-APC-Cy7 (Biolegend); anti-CD14-Pacific Blue (Invitrogen); anti-CD19-Pacific Blue (eBiosciences)). After further washing, PBMC were permeabilised with Cytofix/Cytoperm reagent (BD Biosciences) and stained with an intracellular antibody cocktail (anti-CD3-PerCP (Biolegend); anti-CD8-FITC (Biolegend); anti-IFNγ-PE (Caltag)) prior to a final wash and re-suspension in 1% paraformaldehyde. Cells were acquired within 24 h of staining.

Except where indicated, all reagents were purchased from BD Biosciences. PBMCs were thawed, rested for 10–12 h and viability assessed with Guava ViaCount Reagent (Millipore). Only samples with viability >70% were used for assays. Half a million cells per well were resuspended in RPMI-1640 medium plus 10% fetal bovine serum (culture medium) and incubated with PvLP1 or PvLP2 (5 μg/mL). Culture medium was used as negative control and anti-CD3 as the positive control. After 12 h, an aliquot of 30 μL of culture medium supernatant was collected to measure secreted cytokines, while an equal volume of media containing GolgiPlug was added for additional (4 h) incubation. PBMCs were stained with LIVE/DEAD Fixable Violet Dead (Life Technologies), anti-CD14 Pacific Blue, anti-CD19 Horizon V450, anti-CD4 allophycocyanin (APC) and anti-CD8 Peridinin Chlorophyll Protein Complex (PerCP). After washing, cells were fixed and permeabilized with Cytofix/Cytoperm, and incubated with anti-CD3 phycoerythrin (PE)-Cy7, anti-interferon (IFN)-γ PE and anti-CD69 fluorescein isothiocyanate (FITC). Cells were acquired in a LSRFortessa flow cytometer and data were analyzed by FlowJo (FlowJo LLC, OR, USA). Gating strategy is provided in S2 Fig. Supernatants were frozen at -80°C until Luminex analysis with the Cytokine Magnetic 30-Plex Panel (Invitrogen), according to manufacturer’s instructions.