Cd44 fitc

Cells were rinsed twice with PBS, trypsinized, and centrifuged at 200 ×g for 5 min and then resuspended in 500 mL PBS. Approximately 5 × 10⁵ cells per 100 mL were labeled with primary mouse antibodies against rat CD29, CD90, CD44, CD34, and CD45 at 4°C for 30 min and washed. The labeled cells were analyzed with a BD FASAria Cell Sorter (Beckton Dickinson, San Jose, CA, USA). The antibodies used in this experiment were CD29-FITC, CD90-FITC, CD44-FITC, CD34-FITC, and CD45-FITC (Beckton Dickinson, San Jose, CA, USA). Mouse IgG1-FITC (Beckton Dickinson, San Jose, CA, USA) was used as an isotype control.

The trypsinized GB-MSCs and their respective PBMCs were washed with PBS, centrifuged at 280 g for 10 minutes, counted, and brought to a concentration of 1.10⁵ cells. GB-MSCs were tested for expression of the following surface markers: PD-L1-APC (BD Pharmingen, USA), CD73-PE, CD90-FITC, CD105-PerCP/Cy5-5 (eBioscience, USA), CD45-FITC/CD34-PE, CD44-FITC, CD146-PE, and HLA-A, B, C-FITC (Becton Dickinson, USA) and intracellular markers: Nestin-PE, Sox-2-PerCP, and GFAP-Alexa Fluor 488 (eBioscience, USA). For the detection of intracellularly expressed markers, the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA) was used following the manufacturer's instructions.
PBMCs from healthy volunteers were tested for surface expression of CD3-FITC, CD4-PerCP, CD161-PE, CD196-Alexa Fluor 488, CD25-FITC, CD14-FITC, CD80-PE, CD86-APC, and HLA-DR-PerCP and intracellular expression of FoxP3-PE (BD Pharmingen, USA) by using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA). Cells were processed according to the manufacturer's instructions, fixed with CellFix (BD, USA), and analyzed by FACSCalibur flow cytometer (BD, USA). Software CellQuest and WinMDI 2 were used for further analysis.

Cells were rinsed twice with PBS, trypsinized and centrifugation at 200×g for 5 min, and then resuspended in 500 µL PBS. Approximately 5×10⁵ cells per 100 µL were labeled with primary mouse antibodies against rat CD29, CD34, CD44, CD45 and CD90 at 4°C for 30 min and washed. The labeled cells were analyzed using a flow cytometer (Beckton Dickinson, San Jose, CA, USA). The antibodies used in this experiment were: CD29- FITC, CD34-FITC, CD44- FITC, CD45- FITC and CD90- FITC (Beckton Dickinson, San Jose, CA, USA). Mouse IgG1-FITC (Beckton Dickinson, San Jose, CA, USA) was used as an isotype control.

For the analysis of cell cycle, each culture was plated sparsely at P1 so that it did not touch each other and did not reach contact inhibition. Cells were trypsinized, pelleted and resuspended in 70% ethanol in PBS, and stored at 4°C overnight. Then they were washed with PBS, resuspended in propidium iodide (PI) staining solution (50 mg/ml) and RNAse A (100 Kunitz/ml) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and incubated in the dark for 40 minutes at room temperature.
For the surface antigen expression, the MoAbs used were: CD146-PE, human (Miltenyi Biotec GmbH), CD90.2-APC (Miltenyi Biotec GmbH), CD44-FITC (Becton, Dickinson and Company, Franklin Lakes, New Jersey), CD105-APC (Miltenyi Biotec GmbH) and CD34-PE (Miltenyi Biotec GmbH). Negative control was performed using the HaCaT, spontaneously immortalized human keratinocyte cell line.
For this assay the cells were harvested and stained according to the manufacturer instructions. Each measurement contained at leats 30,000 cells collected with MACSQuant® Analyzer flow cytometer (Miltenyi Biotec GmbH) and analysed with MACSQuantify® software (Miltenyi Biotec GmbH).

Following isolation, the cells were expanded in vitro and then characterized with flow cytometry in order to evaluate the cell surface marker expression at 7, 14 and 21 days of co-culture both with SAOS2 and MCF7 cells. hASCs were characterized with antibodies against the following markers: CD90 FITC, CD34 PE, CD29 PECy5.5, CD44 FITC, CD324 PE and vimentin (from BD Pharmingen, Milan, Italy). For staining, the antibodies were incubated for 30 min at 4 °C. After washing, cells were re-suspended in PBS. For intracellular staining of vimentin, Fix & Perm kit (Invitrogen, Life Technologies Italia) was used following the manufacturer's instructions. All samples were analysed using Diva Software for flow cytometer FACS ARIA III (BD Pharmingen). Statistical evaluation was obtained from three independent experiments. Standard deviation was indicated by error bars.

The BMSCs cell precipitate was collected. After centrifugation, the cells were incubated with CD34-FITC, CD45-FITC, CD11b-FITC, CD44-FITC, CD73-FITC, CD90-FITC, CD105-FITC and IgG-FITC primary antibodies (all from BD Biosciences; USA) for 15 min. Immunophenotype analysis of BMSCs was performed by flow cytometry (BD FACS Canto) [35 (link)].