Splenic B cells were isolated from WT mouse spleens using CD19⁺ magnetic beads. (A) Cells were stimulated with mouse anti-μ (15 μg/ml), LPS, R848, or CpG for 120 hours. Flow cytometry was used at 24-hour intervals to measure GARP⁺LAP⁺ expression on live B cells. Numbers represent the percentage of cells in the GARP⁺LAP⁺ quadrant over the entire CD19⁺ B cell population. Flow plots are representative of 3 independent experiments. A representative baseline flow plot is also shown. (B) Quantification of GARP and LAP expression (n = 4 biological replicates). MFI, mean fluorescent intensity of GARP. Statistical analysis was performed by 2-way ANOVA; ***P < 0.001. (C) Immunoblot of GARP in the whole-cell lysate of untreated (UT) WT B cells or after stimulation with the indicated conditions for 72 hours. Representative of 3 immunoblots. (D) Primary WT and GARP-KO splenic B cells were cultured with LPS, Poly I:C, or IL-1β plus Poly I:C for 72 hours. Cells were stained for GARP and LAP and analyzed by flow cytometry. Representative of 3 independent experiments. (E) Phenotypic analysis of LPS-treated (48 hours) GARP^– and GARP⁺ B cells by flow cytometry. Histogram plots are representative of n = 3 biological repeats and 2 independent experiments. Black lines denote GARP^- cells, red lines denote GARP⁺ cells; shaded areas denote isotype. Numbers represent mean fluorescent intensity (MFI). (F) Human B cells were isolated from normal subjects using human anti-CD19⁺ magnetic beads. Cells were freshly analyzed or cultured with human anti-μ, R848, or CpG for 72 hours. GARP⁺LAP⁺ levels were analyzed by flow cytometry. Representative of 3 independent experiments. (G) Quantification of GARP⁺LAP⁺ expression in 3 biological replicates from healthy donors. Each data point represents an individual donor. Statistical analysis was performed by 2-tailed t test (E) and 1-way ANOVA with Tukey’s multiple comparisons (G); *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SD.