Running buffer

For cytometry analysis, cells were treated with accutase solution (Promocell, Heidelberg, Germany) for 10 min to ensure surface protein integrity. For TrkA staining cells were centrifugated (500 × g, 10 min) and stained immediately with anti-human TrkA PE-conjugated REAfinity antibody (Miltenyi Biotec B.V. & Co. KG) for 10 min in the dark (Table 2). For TrkB staining, cells were incubated after centrifugation with F_C-blocking reagent (Miltenyi Biotec B.V. & Co. KG) for 15 min. Then, cells were incubated with mouse anti-human TrkB Alexa Fluor^® 405-conjugated monoclonal antibody or Alexa Fluor^® 405-conjugated isotype control (IgG₁) antibody (R&D GmbH) for 10 min in the dark at room temperature. Unbound antibodies were removed by washing the cells in 1 mL running buffer (Miltenyi Biotec B.V. & Co. KG) and after centrifugation (500 × g, 10 min), cells were resuspended in 200 μL of running buffer for final flow cytometry analysis. Cell gating strategy and quantification (see Figure 6) were performed using the MACSQuant 2.13.0 software (Miltenyi Biotec B.V. & Co. KG) by comparing the median fluorescence intensities (MFI) of unstained, isotype stained, and Trk-stained cells with n = 3 (each done in duplicates).

After incubation for 24 h, supernatants and cells were collected, and cells were washed. For analyzing the cells’ viability, 4′,6-diamidino-2-phenylindole (DAPI, 1 µM; BioLegend, Amsterdam, The Netherlands) and CD11c conjugated to phycoerythrin (PE) cyanine (Cy) 7 (clone S-HCL-3; BioLegend, Amsterdam, The Netherlands) were added and incubated for 15 min at room temperature in the dark. Subsequently, cells were washed, resuspended in running buffer (Miltenyi Biotec, Bergisch-Gladbach, Germany), and cell data were acquired on a Gallios flow cytometer (Beckman-Coulter, Krefeld, Germany) equipped with an autosampler for FACS tubes. For surface marker analysis, cells were incubated with antibodies (Table 1), washed, and data were acquired on a CytoFLEX LX flow cytometer (Beckman-Coulter, Krefeld, Germany) equipped with an autosampler for 96-well plates. Data analysis was performed using Kaluza 2.1.1 software (Beckman-Coulter, Krefeld, Germany).

OC pieces were digested by 1 mg/mL collagenase and 1 μg/mL DNAse I (Sigma-Aldrich) for 120 min at 37 °C. Cell pellets were then suspended in running buffer (Miltenyi Biotec) and incubated with 10 µL of rabbit anti-human Ddx4 antibody for 30 min at 4 °C. Thus, the samples were treated with anti-rabbit IgG microbeads (Miltenyi Biotec), and Ddx4⁺cells were isolated by an automated magnetic-activated cell sorting system (autoMACS Pro, Miltenyi Biotec,) as previously described [11 (link)]. The purity of cell population was assessed by the flow cytometry detection of membrane Ddx4. Briefly, an aliquot of isolated cells was incubated with rabbit anti-human Ddx4 antibody (Abcam ab13840) and then processed with an FITC-conjugated anti-rabbit antibody (Sigma-Aldrich). In addition, cytoplasmic expression of Ddx4 was also evaluated by flow cytometry, after cell fixation and permeabilization as described for A2780 cells. In each case, an isotype control was used as negative parameter.

Previously fixed samples were centrifuged at 400× g for 10 min to remove PBS. Cells were resuspended in 100 μL of 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) and incubated for 10 min. Samples were centrifuged again to remove BSA before being resuspended in 90 μL of Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). A primary antibody staining cocktail of the following biotinylated antibodies was added and incubated for 15 min at 4 °C: 10 μL of anti-EpCAM (R&D system, Minneapolis, MN, USA), 10 μL of anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), 5 μL of anti-EGFR (RayBiotech, Peachtree Corners, GA, USA) and 20 μL anti-CD45 (BioRad, Hercules, CA, USA). After 15 min, the reaction was quenched with 1 mL of Running Buffer before being centrifuged. The supernatant was discarded and the following solutions were added before incubating an additional 10 min at 4 °C: 90 μL of Inside Stain (Miltenyi Biotec, Bergisch Gladbach, Germany), 2.5 μL of strepavidin Alexa Fluor 488 conjugated (Invitrogen, Waltham, MA, USA), 10 μL of goat anti-mouse IgG2a AF647 (Invitrogen, Waltham, MA, USA), 10 μL of PE anti-pan cytokeratin (Abcam, Cambridge, UK) and 2.5 μL of DAPI (Invitrogen, Waltham, MA, USA). After incubation, the reaction was quenched with 1 mL of Running Buffer and centrifuged.

Cultured PSCs were washed with running buffer (Miltenyl Biotec Inc., Auburn, CA, USA) and centrifuged at 350× g for 5 min (Eppendorf, Westbury, NY, USA). The cells were incubated in blocking solution (Blockaid, Thermo, Austin, TX, USA) at 4 °C for 15 min. PSC samples (1 × 10⁵ cells/100 µL) were incubated with the following antibodies: CD85d-ILT4-PE, HLA-DR- TU36-PE, CD45-HI30-Brilliant Violet, CD73-AD2-PE (StemCell Technologies, Vancouver, BC, Canada), CD90- 5E10-PE (Molecular Probes, Eugene, OR, USA), CD105-43A3-PE, CD273-B7DC-PE, CD119-IFNgRa-PE, CD40-5C3-FITC, CD11b-M1/70-FITC, and CD178-NOK-1-PE (Biolegend, San Diego, CA, USA), and HLAG9-MEM-G/11-FITC (Invitrogen, Austin, TX, USA). Upon completion of the incubation, cells were washed twice with running buffer, centrifuged at 350× g, and then resuspended in 100 µL of running buffer containing 300 mM of DAPI (Biolegend), where they were incubated at 4 °C for 15 min in the dark. Analysis was performed using a Cytoflex flow cytometer (Beckman Coulter, Irving, TX, USA).