Percp anti mouse cd19

Spleen cells were prepared through a 70-mm nylon mesh strainer and washed with PBS. According to a previous reference of Huyan et al. (24 (link)), in short, both spleen suspension and blood sample were stained on ice for 30 min with the following five anti-bodies (Biolegend, San Diego, USA), respectively: PE anti-mouse NK1.1 for natural killer cells analysis, PerCP anti-mouse CD19 for B lymphocyte analysis, APC anti-mouse CD3, FITC anti-mouse CD8a, and Brilliant Violet 421 anti-mouse CD4 for T lymphocyte subset analysis. The percentages of NK cells, B cells, and T-cell subsets were analyzed by a Dxp Athena flow cytometry (Cytek, CA, USA). Ten thousand events were acquired into the list-mode, and staining data were acquired on Flowjo CE software (Tree Star, OR, USA).

The following antibodies were used in the experiments: TruStain Fc block anti-mouse and anti-human CD16/32 (101320; BioLegend), fluorescein isothiocyanate (FITC) anti-mouse CD8 (A502-3B-E; ProImmune), phycoerythrin (PE) anti-mouse CD3e (550353; BD Pharmingen), and peridinin-chlorophyll protein (PerCP) anti-mouse CD19 (115531; BioLegend). SIINFEKL epitope-specific T cells were studied using an APC-labeled H-2Kb/SIINFEKL pentamer (F093-84B-E; ProImmune). Flow cytometric analyses were performed using a BD Accuri 6C Plus Flow Cytometer (BD Biosciences). FlowJo software v.10 (FlowJo) was used for the data analysis.

To acquire single-cell suspension, the mouse spleen was minced through a 40µm nylon cell strainer, and we used the Tris-NH₄Cl lysis buffer (0.017 M Tris-HCl, 0.144 M NH4Cl) to deplete the red blood cells. Then the spleen cells were harvested and divided into two tubes, and 100μL blood was dripped into a tube for testing. One tube of the sample was incubated with the antibody FITC anti-mouse CD3, APC anti-mouse CD4, PerCP anti-mouse CD8, PE anti-mouse CD28, and the other tube of cells were incubated with APC anti-mouse CD4, FITC anti-mouse CD25, and PerCP anti-mouse CD19 (BioLegend) at room temperature for 30 min in the dark before being washed and resuspended in 0.5 mL of PBS and quantified by flow cytometry (BD Calibur™, USA).