Cd24 pe

Peripheral blood mononuclear cells (PBMCs) were acquired and then were stained for surface markers for 20 min in PBS containing fluorescent antibodies. Fluorescently labeled antibodies included CD3-PerCP-Cy5.5, CD25-PE, CD45RA-FITC, CD8-PerCP-Cy5.5, CD19-PerCP-Cy5.5 and CD56-PE-Cy7 (Tianjin Three arrows, China); CD4-APC-H7, CD8-BV510, CD127-BV421, CCR7-AF647, CD28-PE-Cy7, CD3-APC-H7, CD4-PE-Cy7, CXCR3-Alexa488, CXCR6-BV510, CXCR5-Alexa647, CCR4- BV421, PD-1-PE, CD45-APC-H7, CD27-BV421, IgD-BB515, IgM-BV510, CD38-APC, CD24-PE, and CD21-PE-Cy7 (BD, United States). The instrument settings and gating strategies were adopted from previous works (Supplementary Figure S1) (Yang et al., 2020 (link); Zhu et al., 2020 (link)). All experiments, including cell separation and sample preparation, were performed according to standardized experimental manuals. Samples were analyzed using CytoFLEX flow cytometer (Beckman, United States). Results are expressed as the proportion of cells expressing particular markers. T cell subsets, including cytotoxic T (Tc) cells, helper T (Th) cells, T follicular helper (Tfh) cells, regulatory T (Treg) cells, B cells and NK subsets were identified (Supplementary Table S1).

1 × 10⁶ cells were resuspended in 100 μL PBS containing 2% FBS (FACS buffer), and then incubated on ice for 10 min. CD44-APC and CD24-PE (BD Biosciences) were added to the cell suspension and incubated on ice for 30 min. Cells were washed and resuspended in 500 μL FACS buffer and analyzed using a FACS Calibur Flow Cytometer (BD Biosciences).

After 6 days culture, cultured cells were analyzed for expression of surface markers by multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies.
For the analysis of B cell subpopulations and activation markers, the monoclonal antibodies used were CD19-PE-CF594 (BD Biosciences, San Jose, CA), CD62L-PE-Cy5 (BD Biosciences), IgD-FITC (BD Biosciences), CD27-PE-Cy7 (BD Biosciences), CD24-PE (BD Biosciences), CD38-PerCP-Cy5.5 (Beckman Coulter, Brea CA), CD138-PE-Cy7 (eBioscience, San Diego, CA), CD23-PE (BD Biosciences), CD21-PE-Cy5 (BD Biosciences), IgG-PC7 (BD Biosciences), CD86-Alexa 700 (BD Biosciences) and CD95-Pacific Blue (BioLegend, San Diego, CA).
For plasma cell staining, cultured cells were first incubated with monoclonal antibodies to surface markers (CD38, CD138 and CD19), fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) and then incubated with Blimp-1-PE (R&D systems) and Pax5-APC (eBioscience) for 30 minutes.
For the proliferation assay, 1–10×10⁶ cells of interest were labeled with 1μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) before culture initiation. After 6 days of culture, cells were harvested, incubated with antibodies, and dilution of CFSE was assessed by flow cytometry.

Control clones and DEAR1-KD 76N-E6 clones were grown in 2D culture and stained for FACS using EpCAM-PE (eBioscience, 12-9326-41), CD49f-APC (eBioscience, 17-0495-80), CD24-PE (BD Biosciences, 555428), and CD44-FITC (BD Biosciences, 555478) according to the manufacturer’s Staining Cell Surface Antigens for Flow Cytometry protocol. Plated cells were trypsinized, counted, and resuspended in Flow Cytometry Staining Buffer for a final concentration of 1 × 10⁷ cells/mL. 50 µL of the cell suspension was incubated with primary antibody so that the final volume was 100 µL per sample. The cells incubated for 30 min in the dark at 4 °C, washed, and analyzed. For these experiments, 2 stable control vector (CshR) clones and 2 DEAR1-KD (DshR) stable clones were used experiments were performed in triplicate.

After MCF10DCIS cells reached ~80% confluence, they were trypsinized, filtered with a 40 μm cell strainer (Fisher Scientific), and incubated with CD44/FITC (BD Biosciences, Cat No. 555478), CD49f/APC (eBioscience, Cat No. 17-0495-80) or CD24/PE (BD Biosciences, Cat No. 555428) antibodies and 0.5 μg/ml propidium iodide (Sigma) at 4°C for 30 min. CD44⁺/CD49f⁺/CD24⁻ cells that did not take up PI were then sorted into 96-well plates at 1 cell/well using 70 μm nozzle and incubated overnight at 37°C. On day one post-sorting, the plate was monitored under a bright-field microscope equipped with an X-Y stage to confirm that each well contained only one cell. The wells that contained more than one cell or no cells were excluded and the wells containing single cells were monitored everyday.

Single-cell suspensions of each cell line were counted and incubated with antibodies: CD24-PE (BD Pharmingen (ML5; 1:100)), CD44-APC (BD-Pharmingen (G44-26; 1:100)) and EpCAM-FITC (AbD Serotec (VU-1D9; 1:800)). ALDH enzymatic activity was assessed using ALDEFLUOR kit (Stem Cell Technologies) as per the manufacturer's protocol. Diethylaminobenzaldehyde-treated sample served as a negative control for gating. FACS analysis was performed using Becton Dickinson (BD).