Enhanced caspase activity and apoptosis in CD4⁺ T cells after transfection with galectin-10. T cells were resuspended in Nucleofector solution and mixed with 2.5 μg of the galectin-10 expression vector or the empty vector pcDNA 3.1. Cells were immediately nucleofected and resuspended in prewarmed X-VIVO-15. Transfection efficiency was typically around 60% to 65%, controlled by transfection with a GFP-expressing vector and by immunostaining of galectin-10–transfected Jurkat cells. After dead cell removal via Annexin-coupled beads (Miltenyi Biotec), the activities of the active caspase-3 and caspase-7 were measured (Apo-ONE Homogeneous Caspase-3/7 Assay; Promega) as specified by the manufacturer. The amount of fluorescent product generated is representative to the amount of active caspase-3/7 present in the sample. A total of 3 × 10⁵ CD4⁺ T cells per sample were used. After 15 hours, a SpectraFluor reader was used for detection of relative fluorescence intensities. Results are representative of 3 independent experiments; measurements were performed in triplicates (mean ± SD from each triplicate are shown; asterisks indicate P values as defined in “Materials and methods, Statistical analysis”). In addition, cells were stained for Annexin V and PI and analyzed via FACS.