Pe conjugated antimouse cd44

Thymocyte subsets and T-cell subpopulations in blood were investigated by flow-cytometry as published by others (36 (link), 37 (link)). Thymocytes and PBMC were isolated from mice and labeled with fluorophore-conjugated antibodies in PBS-BSA (5% BSA diluted in PBS). In every case, 100,000 cells were stained for measurement. Incubation with antibodies was performed at 4°C for 60 min followed by a washing step. FACSCanto II flow-cytometer and FACSDiva software (Becton Dickinson) were used for analysis. In every case, 10,000 events (parent R1 morphological lymphocyte gate) were recorded by flow-cytometry. For thymocyte subset measurement, Alexa-647 conjugated antimouse CD4 (clone: YTS 191) and FITC-conjugated antimouse CD8 (clone: IBL 3/25) antibodies were used (both produced in the Department of Immunology and Biotechnology, University of Pecs, Hungary). For peripheral blood T-cell subpopulation analysis, Pacific Blue-conjugated antimouse CD3 (clone: 17A2), PerCP-conjugated antimouse CD4 (clone: GK1.5), APC/Cy7-conjugated antimouse CD8 (clone: YTS156.7.7), PE-conjugated antimouse CD44 (clone: IM7), APC-conjugated antimouse CD62L (clone: MEL-14) (all purchased form BioLegend), and FITC-conjugated antimouse CD19 (clone: 1D3, produced by the Department of Immunology and Biotechnology, University of Pecs, Hungary) were used.

The following antibodies were used in flow cytometry: phycoerythrin (PE)-conjugated anti-mouse CD44 (Biolegend, 103007), Sca-I (eBioscience, 12-5981-83), CD140a (Biolegend, 135906), CD13 (BD Pharmingen, 558745), MHC II (eBioscience, 12-5320-80), CD11b (eBioscience,12-0112-83), CD11c (eBioscience, 12-0114-82), CD86 (eBioscience, 12-0862-82), and CD45 (eBioscience, 12-0451-82). Antibodies used for western blotting analysis were: anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology); GAPDH (14C10, 2118, Cell Signaling Technology). Antibody used for Chip was: p53 (FL-393; sc-6243, Santa Cruz Biotechnology), IgG (normal mouse IgG, sc2025, Santa Cruz Biotechnology). The reagents for cell treatment were: Nutlin-3 (Nutlin-3, Selleck Chemicals, Houston, TX, USA), Cisplatin (Sigma-Aldrich), RANKL (recombinant mouse RANKL, 50 ng/ml, R&D), M-CSF (recombinant mouse M-CSF, 25 ng/ml, R&D), and OPG (recombinant OPG, 50 ng/ml, Sigma-Aldrich). RANKL for mouse injection (recombinant mouse RANKL, 2 mg/kg/day) was from R&D Systems.

Flow cytometry analysis was conducted to evaluate the cell surface marker expressions of the CSCs. CSCs with various treatments were harvested and trypsinized into a single cell suspension. Subsequently, cells were incubated with fluorochrome-conjugated antibodies, including FITC-conjugated anti-human cluster of differentiation (CD) 24 (eBioscience, CA, USA), APC-conjugated anti-human CD133 (Biolegend, CA, USA) at 4 °C in the dark. The appropriated isotype-matched antibodies were used as negative controls. After 30-min incubation, the samples were centrifuged and washed with PBS for flow cytometry analysis. Similarly, the cell surface marker expressions of the BMSCs were also evaluated by flow cytometry. The conjugated-specific antibodies used were as follows: PE-conjugated anti-mouse CD14 (Biolegend, CA, USA), APC-conjugated anti-mouse CD34 (Biolegend, CA, USA), APC-conjugated anti-mouse CD45 (Biolegend, CA, USA), PE-conjugated anti-mouse CD44 (Biolegend, CA, USA), PE-conjugated anti-mouse CD73 (Biolegend, CA, USA), APC-conjugated anti-mouse CD105 (Biolegend, CA, USA), FITC-conjugated anti-mouse CD166 (abcam, CA, USA), FITC-conjugated anti-mouse CD29 (Biolegend, CA, USA). Cell analysis was performed using a Flow cytometer (BD Biosciences, CA, USA) and FolwJo software version 7.6 (FlowJo LLC, OR, USA).