Cd44 fitc

Adherent cells from p3 were dissociated and resuspended in flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA) at a final cell concentration of 1 × 10⁶ cells/mL. Human cells were incubated with the following fluorescent monoclonal mouse anti-human antibodies: CD29 APC (Thermo Fisher Scientific, San Diego, CA, USA); CD44 FITC (Thermo Fisher Scientific); CD73 APC (eBioscience^TM, Thermo Fisher Scientific); CD90 BV510 (BD Biosciences, San Jose, CA, USA); CD105 PE-Cyanine7 (eBioscienceTM); CD14 PE (eBioscienceTM); CD34 APC-eFluor 780 (eBioscienceTM); CD45 Pacific Orange (eBioscienceTM). Canine cells were incubated with the following fluorescent monoclonal mouse antibodies: CD29 APC (Thermo Fisher Scientific); CD44 FITC (Thermo Fisher Scientific); CD73 APC (eBioscienceTM); CD90 APC (eBioscienceTM); CD105 PE-Cyanine7 (eBioscienceTM); CD14 PE (Thermo Fisher Scientific); CD34 PE (eBioscienceTM); CD45 FITC (eBioscienceTM). Cells were washed twice with 2 mL of flow cytometry staining buffer and resuspended in 500 μL of flow cytometry staining buffer. Fluorescence was evaluated by flow cytometry in Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using Attune NxT software (Thermo Fisher Scientific).

For staining of CD44 and CD24, cells were exposed to trypsin, washed and suspended in PBS containing pre-conjugated primary antibodies: CD24-PE (1:20, eBioscience, USA); CD44-FITC (1:50, eBioscience) and incubated with the antibodies for 30 min at 4°C. Unstained cells were used for negative control. Cells that only stained with CD24-PE were used to regulate compensation and set CD44-FITC gate, while Cells that only stained with CD44-FITC were used to regulate compensation and set CD24-PE gate. The labeled cells were washed, fixed, and then analyzed with a FACSCalibur Flow Cytometry (BD, USA).
Cells cycle arrest rate was detected by flow cytometry using Cell Cycle Detection Kit (Key-GEN, Nanjing, China). Following its manufacturer's instructions, 2 mL suspension of 10⁶ cells was fixed with 70% ethyl alcohol, and washed by PBS before being stained. The rate of each cycle was analyzed by FACSCalibur Flow Cytometry at 488 nm.
Cells apoptotic rate was also detected by flow cytometry using FITC Annexin V Apoptosis Detection Kit with 7-AAD (Key-GEN, Nanjing, China) according to the manufacturer's instructions. Two mL suspension of 10⁵ cells was stained with (Annexin-V-FITC and 7-AAD) kit solution in dark for 15 min. The apoptosis rate was assayed using FACSCalibur Flow Cytometry at 488 nm.