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. 2015 Dec 3;5:17533. doi: 10.1038/srep17533

Figure 6. In vitro characterization of 3D CTC tumorspheres.

Figure 6

(a) Generation of CTC tumorspheres over time in culture. Tumorsphere assays were performed in FACS sorted (CD45−/CD44+/CD24−/EpCAM-negative/uPAR+/−/int β1+/−) in vitro 3D CTC subsets derived from no BCBM patient. Trypsinized 10-15 3D CTC tumorspheres were cultured in 96-well plate coated with 1% soft agar and quantified at successive weeks under phase contrast microscopy (Zeiss, Inc.); (b) CTCs cell proliferation assays (WST-1, Roche Life Sciences, Inc.) over time in culture were performed in FACS-sorted in vitro 3D CTC subsets containing uPAR/int β1 combinatorial expression. Trypsinized 10-15 3D CTC tumorspheres were cultured in 96-well plate coated with 1% soft agar. Absorbance was measured at 450 nm and 690 nm wavelength at 8 hrs after adding WST-1 reagent at different time points. All data are representative of at least three independent experiments with mean standard deviation (±). Student paired type 2 t-test was performed and p-value* (<0.01) were calculated and found to be significant; (c) CTC adhesion assays. Four CTC subsets with combinatorial expression of uPAR and int β1 were aliquoted into 96 well flat-bottom plates coated with Trevigen® PathClear Basement Membrane Extract® (BME) and incubated for 96 hours at 37 °C for adhesion assay.