Cd34 fitc

Immunofluorescent assay and flow cytometry were employed to detect the phenotypes of hUC-MSCs. Passage 3 hUC-MSCs were seeded into a six-well plate and cultured overnight, fixed by 4% paraformaldehyde for 1 h, and incubated with CD29-FITC, CD44-PE, CD90-FITC, CD105-PE, CD34-FITC, and CD133-PE anti-human antibodies (0.5 μl/well; Abcam, Cambridge, UK) for 1 h at room temperature. After being rinsed three times with PBS, cells were stained with DAPI solution. The fluorescence of hUC-MSCs was detected with the Leica DMR 3000 microscope.
For flow cytometric analysis, passage 3 hUC-MSCs were used to prepare single-cell suspension, and 2 μl of CD29-FITC, CD44-PE, CD90-FITC, CD105-PE, CD34-FITC, and CD133-PE anti-human antibodies were added subsequently into the suspension. These cell suspensions were incubated for 30 min on ice in the dark, and then observed with a BD FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, USA).

The third-generation cultured cells were digested with 0.25% trypsin for 5 minutes. After centrifugation at 1,000 rpm for 10 minutes, the cells were washed twice with PBS, and then the cells were resuspended with PBS to prepare cell suspensions. The cell concentration was adjusted to 2×10⁶/mL. The cells were divided into flow centrifuge tubes at 500 µL per tube, and CD11B-PE (Abcam Cambridge, UK), CD29-PE (Abcam, UK), CD-34-FITC (Abcam, UK), CD45-PE (Abcam, UK), CD90-PE (Abcam, UK), CD105 PE (Abcam, UK), and the isotype control (Abcam, UK) were added. The amount of each added antibody was 10 µL. After incubation at 4 °C for 30 minutes, cells were washed twice with PBS, then 4% paraformaldehyde (Guangzhou Jibeisi Biological Technology Co., Ltd.) at 200 µL was added to each tube to resuspend the cells. A filter membrane was used to filter cell masses to prevent blockage of the flow cytometer. Cells were then fed into the flow cytometer for identification.