Pacific blue conjugated anti cd19

Splenocytes from mice were resuspended in PBS, and Fc receptors were blocked in suspension with human γ-globulin at a final concentration of 0.02 µg/µL (Octapharma) for 20 min at 4ºC. The cells were then incubated with any of the following antibodies in 50 µL of PBS for 20 min at 4ºC: Pacific Blue-conjugated anti-CD19 (Biolegend, 115,523, dilution 1:100), anti-pAkt S473 (Cell Signaling, 4060S, dilution 1:200), anti-pAkt T308 (Cell Signaling, 13038S, dilution 1:200) or anti-pYAP S127 (Cell Signaling, 4911S, dilution 1:200). After being washed with PBS, the cells were incubated with an anti-rabbit TRITC-coupled secondary antibody (Jackson ImmunoResearch, 111-025-003, dilution 1:200) for 20 min at 4ºC. After being washed with PBS, the cells were fixed with paraformaldehyde (2%). Flow cytometry was performed on a CYAN cytometer (Beckman Coulter), and the data were analyzed using FlowJo software, version 10.0 (TreeStar).

Cell suspensions from MLNs were obtained by passing the tissues through a nylon cell strainer with a 40 μm pore size (BD Biosciences) in RPMI1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel). After centrifugation at 300 ×g for 10 min, pelleted MLN cells were suspended in 1 mL of staining buffer. One hundred microliters of cell suspension was incubated with APC-conjugated anti-CD3ε (eBioscience) and Pacific blue-conjugated anti-CD19 (Biolegend) for 30 min at 4°C in the dark. Stained cells were washed and resuspended in staining buffer to measure the lymphocyte population by flow cytometry. Percentages of T and B lymphocytes were determined by CD3ε- and CD19-expressing cells in MLN cells. Representative flow cytometry plots are shown in Figure 2(b).

A five-color flow cytometric analysis was performed to determine the distribution of peripheral blood leukocytes. Antibodies against mouse leukocyte surface antigens were added to 100 μL aliquots of whole blood. The antibodies used to detect different subsets of leukocytes were as follows: PerCP-conjugated anti-CD45 (Biolegend, San Diego, CA, USA) for leukocytes, PE-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA) for monocytes/macrophages, FITC-conjugated anti-Ly6G (BD Biosciences, San Jose, CA, USA) for neutrophils, APC-conjugated anti-CD3ε (eBioscience) for T cells, and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells. Antibodies were used at the concentration recommended by manufacturer. After a 30 min incubation at 4°C in the dark, red blood cells were lysed, and cells were suspended in staining buffer and then analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated, and results are presented as a percentage of specific CD-marker-expressing cells in blood leukocytes. Representative flow cytometry plots are shown in Figure 2(a).