Cd44 apc

Flow cytometry was performed as described previously [47 (link), 48 (link)]. The cells were incubated CD44-APC (BD Pharmingen), however the control were incubated with IgG. After incubation, the cells were suspended in PBS for Flow Cytometry analysis. Mice were sacrificed, and lung tissues were scissors cut into a meaty shape and digested. Cells were stained with fluorescently labeled antibodies. Then cells were analyzed by FACS (BD Biosciences). Finally, the data was analyzed in the Flowjo 7.6.1software. All antibodies information was listed in Supporting Information, Table S2.

MCF-7 and T47D cells were cultured in DMEM-H (HyClone, USA) containing 10% fetal bovine serum (FBS) at 37 °C in a 5% CO₂ incubator. Cells in the logarithmic growth phase, approximately 70–80% confluent, were harvested and digested into a single cell suspension by trypsin digestion.
The complete MammoCult™ medium containing 5% MammoCult™ proliferation supplement, 4 μg/mL heparin, and 0.48 μg/mL hydrocortisone was used. MCF-7 cells were resuspended in complete MammoCult™ medium, and T47D cells resuspended in complete DMEM/F-12 medium. The cell suspension (4 × 10³) was seeded into a 6-well plate and cultured at 37 °C and 5% CO₂. After 7 days of culture, the spheres were collected and centrifuged at 350 g for 5 min, and the supernatant was discarded. Then, 1 mL of Accutase cell dispersion solution was added to digest the spheres, followed by adding 9 mL of sterile PBS solution and centrifugation at 350 g for 5 min. Finally, the supernatant was discarded, and the cells were collected.
CD44⁺CD24^−/low BCSCs were isolated from MCF-7 and T47D cells by staining with CD44-APC, CD24-PE and ESA-FITC (BD Pharmingen, USA) antibodies via FACS as described in our previous research [8 (link), 20 (link)].

Cells were stained with Aldefluor reagent as per manufacturer's instructions (Stemcell Technologies, Cat# 01700) and the percentage of cells with high Aldehyde Dehydrogenase (ALDH) activity were analyzed. Human breast cancer stem cell marker staining was determined by labeling human cells with CD44-APC and CD24-PE antibodies (BD Biosciences, Cat# 559942 and 555428). Dissociated mammary tumor cells and murine cell lines were stained with CD24-PE, CD29-FITC, CD31-APC, TER-119-APC (BD Biosciences, Cat# 553262, 561796, 561814, and 561033) and CD45-APC (Biolegend, Cat# 103111) antibodies, analyzed using the FACS Diva software, and sorted using the Aria Illu sorter (BD Biosciences) [47 (link)].