Beckman cytoflex s system

To explore the development of T cells in thymus, single-cell suspensions in thymus were prepared and subsequently stained with the following anti-mouse antibodies: anti-CD4 (PE-CY7), anti-CD8 (BV510), anti-CD25 (APC) and anti-CD44 (AF700). The proportion of T and B cells in mice was determined by staining splenic cells with anti-mouse antibodies: anti-CD3 (PE) and anti-CD19 (PB450) antibodies (Biolegend). To detect the CD3⁺ T cell subsets, splenic cells were stained with the indicated anti-mouse antibodies: anti-CD3 (PE), anti-CD4 (PE-CY7), anti-CD8 (APC) (Biolegend). To detect T cell percentage in human peripheral blood, anti-human CD3 (FITC), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies were used. All these stained cells were evaluated using Beckman CytoFlex S system (Beckman).

Annexin V/PI and Annexin V/7-AAD Staining Kit (Beyotime) were used to detect cell apoptosis. Splenic cells or peripheral blood in mice were stained with anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) antibodies (Biolegend), as well as Annexin V-PE and 7-AAD. Human peripheral blood was stained with anti-human CD3 (FITC), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-PE and 7-AAD. Purified CD3⁺ T cells obtained from MRL/lpr mice were incubated with DMSO or MLN4924 (0.1 and 0.5 μM) for 12 h and then stained with the indicated antibodies: anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) (Biolegend). Peripheral Blood Mononuclear Cell (PBMC) isolated from patients were treated with 0.5 μM MLN4924 for 6 h and then stained with anti-human CD3 (APC-CY7), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-FITC and PI-PE to detect cell apoptosis. All these cells were analyzed with Beckman CytoFlex S system (Beckman).

The BeyoClick^TM EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime) was employed for the detection of DN T cell proliferation. Mice were intraperitoneally administered with EdU (50 mg/kg) twice a day for 1 week according to previously described methods.^{58 (link)} Then spleen single-cell suspensions were stained with anti-mouse CD3 (APC), anti-mouse CD4 (BV510) and anti-mouse CD19 (PB450) antibodies (Biolegend). At last, the Click-iT reaction was carried out based on the protocols of manufacturer and cells were examined using Beckman CytoFlex S system (Beckman).

To measure T/B cell percentage, single-cell suspensions of spleens were prepared and stained with PE anti-mouse CD3 and FITC anti-mouse CD19 antibodies (Thermo Fisher Scientific) followed by FACS analysis using an FC 500 MC system (Beckman Coulter, Fullerton, CA, USA). To analyze the T cell subsets in spleens, single-cell suspensions were re-stimulated with 50 ng/mL PMA (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 10 μg/mL Brefeldin A (Sigma-Aldrich) for 5 h. Surface markers were stained with the indicated antibodies: FITC anti-mouse CD3, PE anti-mouse CD4, APC anti-mouse CD8, APC anti-mouse CD25 and APC anti-mouse CD4 (Thermo Fisher Scientific). Then cells were fixed with Fixation/permeabilization Buffer (BD Biosciences, San Jose, CA, USA), permeabilized with Perm/Wash buffer (BD Biosciences) and stained with the following antibodies: PE anti-mouse IFN-γ, FITC anti-mouse IL-17, APC anti-mouse IL-4 (Thermo Fisher Scientific) according to the manufacturer’s instructions. For Foxp3 intracellular staining, cells were treated with Foxp3 buffer (BD Biosciences), followed with FITC anti-mouse Foxp3 staining. Stained cells were evaluated using Beckman CytoFlex S system (Beckman).