Anti human cd44 fitc

Cells were harvested with Accutase® (Corning, Catalog # 25-058-CI) and stained with anti-human CD44-FITC (BD Pharmingen), anti-human CD24-PB (Exbio) and anti-human EpCAM-APC (Biolegend) antibodies, as per manufacturer-recommended dilutions for 1 h at room temperature on a rotary shaker. Cells were analyzed in the presence of propidium iodide (1 µg/mL) using a BD LSR Fortessa (BD Biosciences). After doublet discrimination and compensation for spectral overlap, samples were analyzed using FlowJo Software v10.0.7 (BD Biosciences). For sorting, anti-human EpCAM-PerCP/Cy5.5 (Biolegend) antibody was used and cells were sorted using a BD FACS Aria IIu sorter (BD Biosciences).

Doxorubicin hydrochloride (Dox·Hcl) was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, China). Resveratrol (Res) was bought from Guan Jie Biotech (Xian, China). 1-Bromo-4-chlorobutane and triphenylphosphine (TPP) were obtained from Aladdin Co., (Shanghai, China). K₂CO₃ and NaI were supplied by Sinopharm Chemical Reagent Co., (Shanghai, China). Dioleoylphosphatidylethanolamine (DOPE) and hydrogenated soybean phospholipids (HSPC) were purchased from Shanghai A.V.T Technology Co., Ltd. (Shanghai, China). Cholesterol and cholesteryl hemisuccinate (CHEMS) were bought from J&K Scientific Co., Ltd. (Beijing, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), cell mitochondria isolation kit, mitochondrial membrane potential assay kit with JC-1, Caspase 3,8,9 assay kit, ATP assay kit, Lysotracker Green DND and Hoechst 33258 were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). FITC anti-human CD44, FITC Mouse IgG1, PE anti-human CD24 and PE Mouse IgG2a were bought from BD Biosciences (Franklin Lakes, NJ, USA). β-Actin, β-catenin and cyclin D1 primary antibodies and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were provided by Abcam (Cambridge, UK).

Cells were analyzed on a Gallios analyzer (Beckman, Indianapolis, IN, USA) or sorted on a MoFlo Astrios Flow Cytometer (Beckman). Nonviable cells were excluded from further analyses. One million cells were incubated with 5 μLAPC-anti-human CD24 (Biolegend 311118) and 20 μl FITC-anti-human CD44 (BD Pharmingen 555478, San Jose, CA, USA) for 30 min at 4 °C. For CD24-CD44-CD44v6 triple antibody analysis, one million cells were incubated with 5 μl APC-anti-human CD24 (Biolegend 311118), 20 μl PE-anti-human CD44 (BD Pharmingen 555479) and 5 μl FITC-anti-human CD44v6 (R&D FAB3660F, Minneapolis, MN, USA) for 30 min at 4 °C. To detect CD44v7 expression, 500 thousands cells were incubated with 2 μl rabbit anti-human CD44v7 (Millipore AB2083) for 30 min at 4 °C, followed by incubating with 1 μl PE-goat anti-rabbit (Abcam ab97070) for 30 min at 4 °C. The data were analyzed with FlowJov10 (Tree Star, Ashland OR, USA).

The phenotype of MSC was confirmed prior to cell delivery by flow cytometry. Cells suspended in staining buffer containing 1% BSA and 0.05% sodium azide in D-PBS were incubated with Rat BD Fc Block (BD Biosciences, USA) for 5 min., and later with fluorochrome-conjugated antibodies: APC anti-human CD29, FITC anti-human CD44, BV421 anti-human CD90, APC-anti-human CD105, BV421 anti-human CD73 (BD Biosciences, USA), BV570 anti-human CD45 (Biolegend, USA), APC mouse anti-human CD34, APC mouse anti-human CD14 (BD Biosciences) for 40 min. Following washing with a cell staining buffer, cells were fixed with 1% neutral buffered formalin overnight. Samples resuspended in 1% BSA were assessed on the following day using a BD LSR II cell analyzer (Becton Dickinson, USA).

Mammospheres were dispersed to obtain single-cell suspension. Cells were washed in PBS with 2,5% BSA and stained with FITC anti-human CD44 and PE anti-human CD24 (BD Biosciences), according to the supplier's protocol. Flow cytometric analysis was performed on a FACScan and acquisition was performed with WinDI software (Becton Dickinson).