Cd24 alexa 647

Cells were washed in phosphate buffered saline (PBS) and collected by centrifugation. The cells were stained with primary antibodies including CD44-FITC (560977, BD Pharmingen), CD24-Alexa 647 (56144, BD Pharmingen), c-Met-Alexa 647 (566014, BD Pharmingen), CD326-BB515 (565398, BD Pharmingen), CD133-PE (VII 70485, BD Pharmingen) at 4 °C for 30 min. After washing with PBS twice and re-suspended in 800 µL PBS, the cells were filtered through a 70 mm nylon mesh and carried out on a flow cytometer (BD Biosciences, Heidelberg, Germany). The viable and single cells were gated for analyses. BD CellQuest pro (BD Biosciences) and FlowJo (BD Bioscience) software were used to analyze the data.

A single-cell suspension was cultured on 6-well ultralow-attachment plate (Corning) at a density of 4 × 10⁴ cells/well in serum-free DMEM/F12 medium with 1% L-glutamine, 1% penicillin/streptomycin, 2% B27 (Invitrogen), 20 ng/ml EGF (Sigma-Aldrich) and 20 ng/ml FGFb (PeproTech). IL-6 (50 μg/ml) (PeproTech) or tocilizumab (200 μg/ml) was added to examine the mammosphere formation. Mammospheroids were photographed at a magnification of 200× using a Nikon DIAPHOT300 microscope at the indicated time. Mammosphere cells (1 × 10⁵) were further stained with anti-human CD24-PE (BD Pharmingen), anti-human CD44-FITC (BD Pharmingen), CD24-Alexa 647 (BD Pharmingen) or EpCAM-BB515 (BD Pharmingen) for 1 h at 4 °C, PBS rinsed and resuspended in 500 μl PBS. CD44-FITC and EpCAM-BB515 were excited at 490 nm, and the emissions were determined by FL1 PMT (515–545 nm bandpass filter). CD24-Alexa 647 was excited at 633 nm, and the emissions were determined by FL-4 PMT (653–669 nm bandpass filter). BD FACSCalibur flow cytometry (BD Bioscience) and Cell Quest software (BD Biosciences) were used to identify CD44(+)/CD24(−) and EpCAM(+) subpopulations.