Cell surface immunofluorescence staining protocol

To determine the expression of cell surface markers, the cultured cells were stained and analyzed using flow cytometry. Cell staining was performed as per the manufacturer’s cell surface immunofluorescence staining protocol (Biolegend, San Diego, CA, USA). Briefly, the cells were washed twice with PBS and detached using 0.25% trypsin/ethylenediaminetetraacetic acid. The detached cells were collected, washed with PBS, and divided into groups for antibody staining. Each group contained about 1×10⁵ cells for staining. The antibodies used to detect the cell surface markers included CD105, CD90, CD73, CD45, CD34, and CD14 (BioLegend; all antibodies for anti-human). All the antibodies were conjugated with fluorescein dye. The staining was conducted for 30 minutes at 4°C with antibody incubation. After the incubation, the cells were washed twice with PBS. Fluorescence-activated cell sorting (FACS) analysis was performed with a FACS Calibur cytometer (BD Biosciences, San Jose, CA, USA).

Splenocytes were freshly isolated from the spleens of recipient mice. Briefly, the spleen was mashed through a cell strainer and centrifuged at 1000 rpm for 5 min. Then, the cells were washed in cell staining buffer (BioLegend) and centrifuged at 1400 rpm for 5 min. The red blood cells were lysed by ammonium chloride solution (Stemcell) for 10 min, followed by washes and centrifugation. Finally, the cells were stained by FITC anti-mouse CD4 (Catalog #100509) according to the Cell Surface Immunofluorescence Staining Protocol (BioLegend), followed by staining with Alexa Fluor 647 anti-mouse FOXP3 (Catalog #126408) according to True-Nuclear™ Transcription Factor Staining Protocol (BioLegend). After that, the percentage of CD4⁺ Foxp3⁺Treg cells was evaluated by flow cytometry.