Clone g44 26

DLD-1 parental and KOOKi-67 clones were subjected to flow cytometry analysis for the expression of CD44 and CD133. Briefly, 2 million cells were harvested from 85% confluent flasks and resuspended in PBS with 0.1% BSA. The cells were washed and incubated at 4°C for 15 minutes with anti-CD44-allophycocyanin (APC) (1:20 dilution, clone G44-26, BD Biosciences) and anti-CD133-phycoerythrin (PE) (1:20 dilution, clone AC133, Miltenyi Biotec) antibodies, or mouse-specific IgG_2b ĸ-APC (1:100 dilution, BD Biosciences) and IgG₁-PE (1:20 dilution, Miltnyi Biotec) antibodies. The cells were then washed and resuspended in PBS with 0.1% BSA and 2μg/mL propidium iodide (PI), and a FACSCalibur flow cytometer (BD Biosciences) was used for all analyses. The cells were first gated on the basis of side-scatter and forward-scatter, followed by the exclusion of nonviable (PI-positive) cells. The CD44⁺ and CD133+ gates were created on the basis of cellular staining with the isotype control antibodies (IgG_2b ĸ-APC and IgG₁-PE, respectively).

AF cell cultures at P0 from fetuses with spina bifida aperta (isolated as described above), grown in 10 cm petri dishes for 5–7 days to 70–80% confluency, were detached with Accutase (StemCell Technologies, Vancouver, BC, Canada) and resuspended in staining buffer (PBS supplemented with 0.5% BSA (w/v) and 2 mM EDTA). Cells were stained with primary antibodies for 30 min at 4 °C in staining buffer: anti-human CD29-Alexa Fluor 488 antibody (1:50, clone TS2/16, BioLegend), anti-human CD73-PE antibody (1:50, clone AD2, BD Biosciences, Eysins, Switzerland), anti-human CD44-PE antibody (1:50, clone G44-26, BD Biosciences), anti-human CD90-FITC (1:50, clone 5E10, BioLegend) and anti-human CD105-Alexa Fluor 488 antibody (1:50, clone 43A3, BioLegend). Samples were analyzed on a BD LSRFortessa flow cytometer equipped with the FACSDiva Software at the Cytometry Facility of the University of Zurich. Data were analyzed using FlowJo (BD Life Sciences, Ashland, OR, USA).

1 × 10⁶ cells of each MCF-7-LeGO and -Δ40p53 sublines were resuspended in 100 μL of ice-cold 2% FBS in PBS containing allophycocyanin (APC)-conjugated mouse-anti-human CD44 (20 μL; clone G44-26; BD Biosciences, Becton Dickinson Pty Ltd, Macquarie Park, NSW, Australia #550392), BD Horizon Brilliant Violet 421 (BV421)-conjugated mouse anti-human CD24 monoclonal antibody (20 μL; clone ML5; BD Biosciences #562789), and 7-amino-actinomycin D (7-AAD) for 20 min at 4 °C in the dark. The cells were then rinsed twice with 2% FBS in PBS. CD44 and CD24 levels were determined using a BD FACS Aria III flow cytometer (BD Biosciences). Unstained cells were used as negative controls.