Anti epcam fitc

Single-cell suspensions generated in PBS supplemented with 1% FBS were incubated with anti-EpCAM-FITC (1.20, Genetex, GTX30708), and anti-CD44-APC (1.20, BD Pharmingen, 559250) antibodies for 30 min on ice and analyzed on a FACSAria III Cell Sorter (BD Biosciences). CD44^hiEpCAM^hiand CD44^hiEpCAM^lo HCT116 and SW480 cells were sorted and cultured in humidified atmosphere at 37°C with 5% CO₂ for 3–5 days before collecting RNA or protein, as previously described (Sacchetti et al., 2021 (link)). The subpopulation of cells mapping in between the CD44^hiEpCAM^hi and CD44^hiEpCAM^lo gates was labelled as intermediate and was further not employed for analysis.

Single-cell suspensions in PBS supplemented with 1% FBS were incubated with anti-EpCAM-FITC (1:20, #GTX30708, Genetex), and anti-CD44-APC (1:20, #559250, BD Pharmingen) antibodies for 30 min on ice and analyzed on a FACSAria III Cell Sorter (BD Biosciences). CD44^hiEpCAM^hi and CD44^hiEpCAM^lo OV90 and CAOV3 cells were sorted and incubated in a humidified atmosphere at 37 °C with 5% CO₂ for 3–5 days before RNA or protein were collected as described above. See Fig. 1 for a more detailed protocol and gating specifications.
Patient-derived ascites were first washed 1-2 times with 1 $\times$ RBC lysis buffer [150 mM NH₄Cl (#7173-51-5, Sigma Aldrich), 10 mM KHCO₃ (#298-14-6, Sigma Aldrich), 100 μM EDTA (#60-00-4, Sigma Aldrich)] to remove erythrocytes. The cell pellets were then labeled with anti-CD90 (Brilliant® Violet 421; 1:20, #328122, Clone:5E10, BioLegend), anti-CD45-APC (1:20, #304037, Clone: HI30, BioLegend), and SYTOX^TM Red (1:1000, #S34859, Thermo Fisher Scientific) antibodies, and sorted by FACS. RNA from CD45^-CD90^- and CD45^-CD90⁺ cells was isolated directly after FACS sorting.