Countless 2

The cell proliferation assay was performed as previously described [14 (link)]. Briefly, 2 × 10⁶ THP-1 cells/well were incubated with ATC cell-derived CM or DMEM (control) in six-well plates, with each assay carried out in triplicate. The number of viable cells was counted 24 h and 48 h after treatment using a cell counter (Countless II, ThermoFisher Scientific, Waltham, MA, USA).

To assess cell viability in low attachment conditions, 250,000 MCF-7 cells were seeded in 6-well low attachment plates (Corning, NY, USA). Forty-eight hours later, cell viability was assessed using trypan-blue staining. Non-viable cells were excluded and living cells were quantified using an automatic counter (Countless II, ThermoFisher, Waltham, MA, USA). Cells were reseeded in 96 well plates and cell viability was assessed 72 hours later using the CellTiter-Blue Assay (Promega), as described^{41 (link)}. Absorbance was detected (560 nm excitation and 590 nm emission) using a Victor X4 Multimode Plate Reader (PerkinElmer, Hopkinton, MA, USA). Experiments were performed in triplicates in at least 3 independent experiments.

Single-cell suspensions were prepared from BALFs, and total cells in BALFs were counted by Countless II (ThermoFisher). Cellular differential percentages in BALFs were measured by flow cytometry on an Accuri C6 Plus Flow Cytometer (BD Biosystems), and the data were analyzed with FlowJo software (Tree Star Inc.) as previously described (38 (link)).