Luminex 200 instrument

Cells were seeded at a density of 1 × 10⁵ cells/cm² and infected with Tha or Th2P-4M at an MOI of 5. Forty-eight hours post-infection, cells were lysed using Procartaplex™ cell lysis buffer (EPX-99999-000, Thermo Scientific) according to the manufacturer’s instructions to quantify protein expression in the cellular cytoplasm. Intracellular protein concentrations of human IFN-β, IFN-γ, IL-1β, IL-6, IL-15, CXCL10, LIF, CCL5, and TNF-α were quantified using a 9-plex Procartaplex assay (PPX-09, Thermo Scientific). In short, DropArray 96-well-plates (96-CC-BD-05, Clinisciences, Nanterre, France) were blocked using 1% BSA for 30 min. After blocking, 20 µL of cell lysate was added per well. According to the protocol, plates were stepwise incubated with 5 µL detection antibody, 10 µL streptavidin-PE, and 10 µL reading buffer per well before being read by the Luminex 200™ instrument (Thermo Scientific).

Serum collected at time of necropsy was stored at − 80 °C until analysed. Samples from all gestation (GD11 and GD18) and treatment (control and infected) groups were analysed at the same time within the same assay. Briefly, 50 µL of serum were analyzed for IL-1α, G-CSF, IL-10, IL-17A, IL-1β, IL-6, TNFα, IL-4, GM-CSF, IFNγ, IL-2, IL-5, IL-13, IL-12p70, Eotaxin, GRO-α, IP-10, MCP-1, MCP-3, MIP-1α, MIP-2, and Rantes (Rat Cytokine & Chemokine 22-plex ProcartaPlex Panel, Invitrogen, Catalog #EPX220-30122-901) as per the manufacturer’s instructions. Samples were analysed with a Luminex 200 instrument (Thermo Fisher Scientific, Waltham MA).

Blood was withdrawn from mice under terminal anaesthesia by cardiac puncture using a needle rinsed with heparin and transported on ice for centrifugation. Plasma samples were then stored at -80°C before downstream analysis. Cytokine and chemokine concentrations in the supernatant (pg/mL) were measured using MILLIPLEX mouse 16-plex (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-21, IL-22, IL-23, IL-27, GM-CSF, IFNγ, TNFα), IL-18 single plex and TGFβ1 single plex magnetic bead kits (Milli-pore, MA). The plate was prepared as per the protocol described i n[80 ] and was read on a Luminex 200 instrument (Thermo Fisher) at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM), Oxford.

Luminex Multiplex kits (R&D Systems, Bio-Techne, Minneapolis, MN, USA) were used for the simultaneous quantification of selected human cytokines, chemokines and growth factors: IL-1β, IL-6, IL-8, IL-10, Growth/Differentiation Factor (GDF)-15, HGF, MCP-1 and VEGF in cell supernatants collected previously and frozen in −80 °C (preparation procedure describe above). The analysis was performed according to the manufacturer’s specification and analysed by the Luminex 200 Instrument (Thermo Fisher Scientific, Waltham, MA, USA). The experiment was performed in duplicates on cell populations from 6 donors.

RA synovial tissues from five donors were disaggregated as above, stained with antibodies to CD45 (2D1), CD31 (WM59), PDPN (NZ-1.3), THY1 (5E10), CD34 (581) and Zombie NIR (BioLegend) for fluorescence-activated cell sorting (BD FACSAria III Cell Sorter). FLS populations (CD45⁻CD31⁻PDPN⁺THY1⁻CD34⁻ versus CD45⁻CD31⁻PDPN⁺CD34⁺) were collected directly into cell culture medium and plated at 20,000 cells per well in a 96-well plate. After adhering for 30 min, they were stimulated for 24 h with TNF (20 ng ml⁻¹), IFN-γ (100 U ml⁻¹) and IL-1β (1 ng ml⁻¹) or a medium-only control. Supernatant was collected, centrifuged and assayed via a custom ProcartaPlex assay (Thermo Fisher Scientific) on a Luminex 200 Instrument. The cells were collected in trizol for bulk RNA sequencing.