Anti cd24 fitc

To identify the expression of CD44/CD24 markers from the MCF7_DoxS and MCF7_DoxR cells, a flow cytometry assay was performed. Both MCF7_DoxS and MCF7_DoxR cells were detached using StemProAccutase^® Cell Dissociation Reagent (Gibco, Waltham, MA, USA). MCF7_DoxS and MCF7_DoxR cells were washed twice with PBS (Gibco, Waltham, MA, USA) and 1 × 10⁶ cells/mL from each cell type were incubated with anti-CD44-PE (Biolegend, San Diego, CA, USA) and anti-CD24-FITC (Biolegend, San Diego, CA, USA) for 20 min in the dark at room temperature. Following that, cells were centrifuged at 300× g for 5 min and re-suspended in 300 µL PBS. PE- and FITC-IgG1 isotype controls (Biolegend, San Diego, CA, USA) were used for the nonspecific binding. The expression profiles were analyzed by a Fluorescein activated sorter FACS Canto II using FACS Diva 7 software (BD San Diego, CA, USA).

Anti-CD44 and anti-CD24 flow-cytometry was conducted to quantify CSC-like cells. Cancer cells were harvested and counted to prepare the appropriate number of cells for flow analysis. A total of 1 × 10⁶ cells/mL of the single-cell suspension were suspended in 100 µL of phosphate-buffered saline (PBS), followed by double staining with a 1:40 dilution of anti-CD24-FITC (Biolegend, San Diego, CA, USA) and anti-CD44-PE ((Biolegend, San Diego, CA, USA) in PBS. The reaction was incubated at 4 °C for at least 1 h in the dark. The cells were then washed with PBS to remove unbound dyes. The stained cell pellet was resuspended in 500 µL of fresh PBS, then subjected to a BD Accuri C6 flow cytometer and analyzed using BD Accuri C6 software (BD, Franklin Lakes, NJ, USA). Gating of the targeted CD44⁺ and CD24⁺ population was based on the control cells, incubated with isotype control mouse IgG2a-FITC and rat IgG2a-PE.

WT/PyMT and 211F/PyMT cells were generated as described previously [18 (link)]. PyMT-derived cells, T47D and BT474 cells were cultured in DMEM/F12 with 10% FBS and 1% penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Anti-SMA and anti-plakoglobin antibodies were purchased from Abcam (Cambridge, UK); anti-vimentin and anti-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA). Anti-E-cadherin and anti-N-cadherin antibodies were purchased from GeneTex (Irvine, CA, USA). Anti-CD24-FITC and CD29-PE antibodies for flow cytometry were purchased from Biolegend (San Diego, CA, USA). Mouse CD326 MicroBeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Collagenase type IV was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Antibodies for flow cytometric analysis included anti-CD24-FITC (Biolegend), anti-EpCAM-FITC (Stemcell Technologies), anti-CD49f-PE/Cy5 (BD bioscience), and biotinylated anti-CD31/CD45/CD235a (Biolegend). Pacific Blue-conjugated streptavidin (Invitrogen) was used to visualize the biotinylated antibody cocktail. Antibodies used for IHC/ICC include CK5 (Abcam 1:1000), CK8 (Abcam 1:500), CK14 (Abcam 1:200), CK18 (Abcam 1:200), CD10 (Abcam 1:50), SMA (Abcam 1:200), and ESA (Leica 1:200).