Pe labeled mouse anti human cd24

Flow cytometry was applied to compare the expression of a panel of CSC markers, some of which are known renal CSC markers, including CD24, CD44, CD133, CD105, CXCR4, and CD73 (23 (link)). Other cell surface markers such as CD29, CD34, CD56 (NCAM), CD90, CD117, and CD146 have been found to be differentially expressed between PCs and SDCs of other cancers (24 (link)). The SDCs were dissociated by accutase and then washed with PBS. Nearly 1 × 10⁵ single viable cells from each population were incubated with 1 μg/ml fluorescently labeled monoclonal antibodies, or respective isotype controls, in the dark for 30 min on ice. Several antibodies were used: phycoerythrin (PE)-labeled mouse anti-human CD24, PE-labeled mouse anti-human CD29, FITC-labeled mouse anti-human CD34, fluorescein iso thiocyanate (FITC)-labeled mouse anti-human CD44, PE-labeled mouse anti-human CD56, PE-labeled mouse anti-human CD73, FITC-labeled mouse anti-human CD90, PE-labeled mouse anti-human CD105, PE-labeled mouse anti-human CD117, PE-labeled mouse anti-human CD133, PE-labeled mouse anti-human CD146, and PE-labeled mouse anti-human CXCR4 (all, BD Biosciences, USA, except CD90, DAKO, Denmark). Then, the cells were washed, resuspended, and analyzed by flow cytometry (FACS Calibur, BD, USA). The FlowJo Version 7 was also used to analyze the data.

Approximately 48 h post-transfection, miR-125b.pBUD transfected MCF-7 cells were treated with 4% paraformaldehyde for 30 min at 25 °C. Permeabilization of the cells were achieved through incubating with 0.4% Triton X-100 for 20 min. After cell washing, primary antibody was diluted by blocking solution (BSA, 5 mg/mL) and used for cell staining. The cells were then incubated at 4 °C overnight. In the next step, the cells were stained with secondary antibody for 1 h at 37 °C and finally, they were stained with DAPI (D9542, Sigma) at final concentration of 1 μg/mL at room temperature for 5 min. Both first and secondary antibodies were Musashi-1 antibody (1:100, R&D: AF2628) and goat anti-rabbit IgG-FITC (1:100, Santa Cruz: Sc-2012). Images were captured under a fluorescent microscope (Olympus, Center Valley, PA, USA) using Olympus DP70 camera. CD24 expression was assayed using flow cytometry. Co-transfected MCF-7 cells with miR-125b.pBUD and pBUD.USc were detached using triple (Gibco, 12,604–012) after 48 h. Treated cells were stained with PE labeled mouse Anti-Human CD24 (BD Pharmingen™, 555,428, USA) for 30 min. After washing, the cells were evaluated by FACS Caliber flow cytometer (Becton Dickinson, USA and obtained results were analyzed with the ModFit LT™ v 4.0 program.