Facs accuri c6 plus

Cells (2 × 10⁶ cells per sample) were incubated with indicated compounds for 24 h. For harvesting, cells were trypsinized and briefly washed with ice-cold PBS twice. Samples were stained with Alexa Fluor 488 conjugated annexin V (A13201, Thermo Fisher, Waltham, MA, USA) and propidium iodide (#556463, BD Biosciences, Bedford, MA, USA). To eliminate the debris and prevent false-positive or -negative results, unstained cells were excepted by gating. Thereafter, apoptotic cells were analyzed by FACS Accuri™ C6 Plus (BD Biosciences, Bedford, MA, USA).

Cells were washed with Straining buffer (Invitrogen) twice and re-suspended with 500 μl Straining buffer. Then cells were incubated with 5 μl CD133 antibody (Invitrogen, 17-1338-42, USA) or EPCAM antibody (Invitrogen, 12-9326-42, USA) on ice for 40 min in dark. Subsequently, cells were re-suspended with a 500 μl Straining buffer (Invitrogen) and tested on the FACS Accuri C6 PLUS (BD).

Apoptosis analysis was evaluated by staining cells with Annexin V‐FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) or Annexin-V‐APC (BD Pharmingen, le Pont de Claix, France) for coculture experiments according to manufacturer’s instructions. For cell cycle analysis, cells were fixed with ethanol 70% for 1 h, and stained with propidium iodide (10 µg.ml⁻¹). Flow cytometry analysis was performed on FACS Accuri C6 plus (BD Biosciences). All experiments were repeated at least 3 times.

The placental samples were homogenized by mechanical disaggregation of tissues using the Medimachine System (120V; BD™, San Jose, CA, United States). The samples were cut in small pieces, which were inserted into a Medicon of 35 μm (BD™ Medimachine Medicon, Sterile) by adding 10 μl protease-free PBS (Sigma, St. Louis, MO, United States) for 3 cycles at 80 rpm. The homogenized samples were passed through Filcons of 20 μm (BD™ Medimachine Filcon, Sterile, Cup-Type), a washing cycle at 1800 rpm for 5 min was done, and the supernatants were recovered to measure the soluble cytokine levels using a multiplex bead-based LEGENDplex™ assay by flow cytometry. A Human Th Cytokine mix and match 13 plex Panel (San Diego, CA, United States) was customized, in which 13 capture beads of two different sizes were used, each one conjugated with antibodies against IFN-γ, IL-12p70, IL-17A, IL-17F, IL-23, IL-21, IL-10, IL-15, granzyme B, MCP-1, TNF-α, IL-1ß, or IL-6. The samples were processed according to manufacture instructions in a sandwich immunoassay. The flow cytometry acquisition was carried out in a BD FACS Accuri™ C6 Plus using the BD Accuri™ C6 Plus software; the analysis was performed based on a standard curve into a logistic regression model (five points; BioLegend’s LEGENDplex™ Software, 2016). The results were expressed in pg/ml units.

The supernatants of both the 2D and 3D cell cultures in the different experimental conditions were collected. The levels of the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α and the chemokines RANTES, MCP-1α, MIP-1α, and IL-8 were measured by flow cytometry with a LEGENDplex™ kit (BioLegend, San Diego, CA, USA). The data acquisition was performed with a BD FACS Accuri™ C6 Plus, and the data processing was performed using LEGENDplex software v8.0 (Biolegend, San Diego, CA, USA). Each experimental condition was analyzed in duplicate for three independent experiments.

The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).