Fitc anti human cd90

Detailed procedure can be found in our previously published study [89 (link)]. Briefly, hMSCs at P4 passage were collected and centrifuged at 1000 rpm for five minutes. The pellet was resuspended in 3 ml/well wash buffer (98% PBS + 2% Fetal Calf Serum) and counted with Countess Automated Cell Counter (C10227, Life Technologies). About 4.5 × 10⁵ cells in 100 μl were aliquoted into each FACS tube (coated with 1% BSA overnight at 4 °C prior), and 5 μl of each labeled primary antibody was added in each tube for staining for 30 min at 4 °C. Cells were stained with FITC anti-human CD90 (cat# 328107, Biolegend) alone, Pacific Blue anti-human CD73 (cat# 344011, Biolegend) alone, or both together for 30 min at 4 °C. FITC Mouse IgG1 (cat# 400109, Biolegend) was used as isotype controls. Unstained hMSCs were also used as negative controls. Cells were then fixed with 2% paraformaldehyde for 30 min at RT, washed with PBS once before flow cytometry analysis. Flow cytometry data was acquired through a Gallios flow cytometer (Beckman Coulter) at the City of Hope Analytical cytometry core and analyzed using the FlowJo software by Tree Star Inc.

The marker expression of BM-hMSCs unstimulated or stimulated using 1.3 nM rhHGF or 32 nM aMD4dY-PA22 for 3 days were analyzed via flow cytometry. Harvested cells were blocked with PBS supplemented with Human TruStain FcX (Biolegend) for 10 min on ice. FITC-anti-human CD90 (Biolegend), PE anti-human CD105 (Biolegend), PE anti-human CD34 (Biolegend), FITC-anti-human CD45 (Biolegend), or FITC-anti-CD73 (Biolegend) diluted 10 times with a cell stain buffer (PBS supplemented with 1% FBS) was added to cells and incubated for 1 h on ice. For the isotype controls, 1:10 diluted PE mouse IgG1 k Isotype (Biolegend) and FITC mouse IgG1 k Isotype (Biolegend) were utilized. After washing two times with a cell stain buffer, cells were analyzed using Attune NxT flow cytometer (Thermo Fisher Scientific).

The expression of typical surface markers in PDLSCs at the third passage was analyzed using a Novocyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA). The adherent cells were collected and resuspended in 50 μL of phosphate-buffered saline (PBS). The harvested cells were stained with 5 μL antibodies, namely FITC anti-human CD31 (#303103, BioLegend, San Diego, CA, USA), FITC anti-human CD34 (#343603, BioLegend), FITC anti-human CD73 (#344016, BioLegend), FITC anti-human CD90 (#328107, BioLegend), or FITC anti-human CD146 (#361011, BioLegend), in the dark for 30 min at 4 °C. Raw data were analyzed using the FlowJo software (FlowJo, Ashland, OR, USA).