Pe cy 7 mouse anti human cd24

Cells were harvested, washed, and numbers adjusted to 1 × 10⁵ to 5 × 10⁵ cells/mL in ice cold FACS buffer. 100 μL of cell suspension was stained in polystyrene round‐bottom 12 × 75 mm BD Falcon tubes with 0.1–10 μg/mL of the primary antibodies and incubated 30 minutes at 4°C in the dark. After washing, cells were analyzed using a LSRF Fortessa flow cytometer (BD Biosciences, Mississauga, ON, Canada). Antibodies used were: PE‐Cy 7 Mouse Anti‐Human CD24 (BD Pharmingen, Mississauga, ON, Canada; Cat #561646), Alexa Fluor 700 Mouse Anti‐Human CD44 (BD Pharmingen; Cat 561289), and CD49f (Integrin alpha 6) FITC Monoclonal Antibody (eBioscience; Cat #eBioGoH3).

After a 72 h treatment, SUM159 and MCF10A cultures were trypsinized, counted, washed with phosphate-buffered saline (PBS) and stained with cell surface markers: FITC anti-human CD326 (EpCAM) (BioLegend, Inc., San Diego, CA), PE-Cy7 Mouse Anti-Human CD24 (BD Biosciences, San Jose, CA), and APC Mouse Anti-Human CD44 (BD Biosciences, San Jose, CA). One million cells were incubated with the antibodies for 15 minutes in dark on ice with manufacturer recommended concentrations. After washing with PBS, cells were analyzed using the LSRFortessa cell analyzer (BD Biosciences, San Jose, CA). Side and forward scatter were used to eliminate debris and cell doublets. Unstained and IgG2a, κ isotypes with PE-Cy7, FITC, and PE (BD Biosciences, San Jose, CA) stained samples were used as controls.

CSC populations (CD24⁻/CD44⁺ and ALDH⁺) were measured by flow-cytometry of single-cell suspensions obtained from tumor biopsies (Additional file 1: Supplemental Materials) by using anti-human CD44-APC (clone G44-26) and CD24-FITC (clone ML5, RUO) antibodies (BD Biosciences, Franklin Lakes, NJ), and ALDEFLUOR assay (StemCell Technologies, Inc., Vancouver, BC, Canada), respectively, as previously published [13 (link)].
Additional analyses were directed to determine CXCR1 (PerCP/Cy5.5 mouse anti-human CD181 (CXCR1; BioLegend, San Diego, CA) and cell lineages using the following antibodies: APC mouse anti-human CD44 (BD Bioscience, San Jose, CA; catalog #559942), PE-Cy7 mouse anti-human CD24 (#561646), PE mouse anti-human CD2 (#555327), PE mouse anti-human CD31 (#555446), PE mouse anti-human CD3 (#555333), PE mouse anti-human CD18 (#555924), PE mouse anti-human CD16 (#555407), PE mouse anti-human CD19 (#555413), and PE mouse anti-human CD45 (#555483).