Anti mouse cd19 bv650

Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Single cell suspensions from spleens of un-immunized C57BL/6 mice were prepared as described previously and incubated with supernatant containing TA99, TA99-Neo-2/15, TA99-HL2-KOA1, or TA99-IL2 at indicated concentrations for 30 min at 37°C. Following stimulation, cells were washed and incubated with Live/Dead Fixable Aqua Dye, anti-mouse CD4-BV421, anti-mouse CD19-BV650, anti-mouse CD25-FITC (all from Biolegend) for 10 min at room temperature. The cells were then fixed using BD Cytofix (BD) and permeabilized using Perm Buffer III (BD) according to manufacturer’s protocol. Cells were then stained with anti-mouse pSTAT5-PE (ThermoFisher) for 30 min at room temperature. The cells were then resuspended in FACS buffer and acquired with a BD FACSymphony A3 flow cytometer.