Fitc mouse anti human cd90 clone 5e10

Two million patient-matched CAF or NPF were co-stained with FITC mouse anti-human CD90 (clone 5E10; BD Biosciences, San Diego, CA) and PE anti-human CD166 (clone 3A6; Biolegend) for 20 min on ice in FACS Buffer (PBS containing 10% HI-FBS and 5 mm EDTA). Cells were washed with PBS and re-suspended in 200 μl of FACS buffer containing 1 μg/ml propidium iodide (PI) to label dead cells. 3 × 10⁵ live cell events were collected on the LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software v10 (BD Biosciences). Statistical significance was determined using a Mann-Whitney U test (p value <0.05).

A poly(2-hydroxethyl methacrylate) (polyHEMA) coating was prepared as described elsewhere [50 (link)]. In brief, the PolyHEMA solution was poured into the wells of a 24-well tissue culture polystyrene plate to cover the surface. The plate was then air dried in a laminar airflow chamber overnight. The MSCs, MDA-MB-231 and MCF7 cells were seeded at a density 5 × 10⁴ cells per well on polyHEMA-coated plates in the complete cell culture media. Then, the 3D spheroid formation was analysed using an EVOS XL light transmission microscope at 24, 48 and 72 h (AMG, Washington, USA).
To distinguish between cell populations in the co-culture, CD90 was chosen as a selective marker for MSCs and EpCAM was chosen as a marker for MCF7 cells. CD90 expression dynamics were analysed in MSCs after 24, 48, 72 and 96 h of propagation in 3D culture. The spheroids were pelleted by centrifugation at 250 g for 5 min, trypsinised for 5 min at 37 °C to obtain a single cell suspension, and finally centrifuged and suspended in 100 μL of PBS. The samples were stained with FITC mouse anti-human CD90 (clone 5E10, BD Bioscience, Carlsbad, CA, USA) or FITC mouse anti-human EpCAM (clone EBA-1, BD Bioscience, Carlsbad, CA, USA) for 30 min at room temperature. Flow cytometry data were acquired on a Guava EasyCyte 8HT flow cytometer and analysed by ExpressPro software (Millipore, MA, USA).