Cls3814

Cells were plated as single cells at a density of 5 × 10² viable cells/well in ultralow attachment six-well plates (Corning, CLS3814). Cells were grown in a serum-free DMEM or phenol-red free DMEM, supplemented with B27 (Invitrogen, 17504-044), 20 ng/mL EGF (Sigma, E9644) and 20 ng/mL bFGF (R&D systems, 233-FB-025). Mammospheres were grown for 10–14 days, and phase contrast images were obtained using the ImageXpress Micro microscope (Molecular Devices). For the second-generation experiment, first-generation mammospheres were collected from multiple wells and spun at 500 × g per 5 min. The pellet was resuspended in 50 μl of Trypsin, and the sample was passed 25 times through a sterile needle to get single-cell suspension. The same density of cells as in first-generation culture was seeded, and cells were allowed to grow for 14 days.

CM61 cells were cultured either as adherent cultures or on low attachment plates (Corning Cat#CLS3814), following which they were either scraped or pelleted to isolate cells for surface proteome preps. Then, 4 × 10⁷ cells were taken for each prep. The CM61 CTC cell line was grown in low attachment plates. The cell line was subsequently mixed with naïve neutrophils in a 1:1 ratio in HBSS medium (Thermo Cat#14185052) for 1 h; the cells were then pelleted and subjected to surface proteome preps. The Pierce Cell Surface Protein Isolation kit (Thermo Cat#89881) was used for all cell proteome labeling and isolation. The media were removed and cells were washed twice with ice-cold PBS. Surface proteins were cross linked with Sulpho-NHS-SS-Biotin in ice-cold PBS for 30 min at 4 °C. The reaction was quenched and washed with TBS twice. Cell lysis was performed by lysis buffer provided in the kit and cells were sonicated on ice using five one-second bursts. Cells were then incubated on ice for 30 min and vortexed every 5 min for 5 sections. Samples were then centrifuged at 10,000 g for 2 min at 4 °C. The supernatant was incubated with NeutrAvidin agarose slurry and incubated for 60 min with end-over-end mixing using a rotator. The beads were then washed twice, after which they were eluted with a buffer containing 8 M urea and 50 mM DTT.