Tim-3 recognizes apoptotic cells. (A) NRK cells stably expressing Tim-1, Tim-2, Tim-3, or Tim-4 were stained with biotinylated anti–Tim-1 mAb (RMT1-17), anti–Tim-2 mAb (RMT2-14), anti–Tim-3 mAb (RMT3-23), or anti–Tim-4 mAb (RMT4-54), respectively. Parental NRK cells were stained with a cocktail of all mAbs. Then cells were stained with PE-avidin, and analyzed by flow cytometry (thick histogram). Thin histograms indicate background staining with control rat IgG2a, followed by PE-avidin. (B) These NRK cells were cultured with CFSE-labeled apoptotic cells for 30 minutes at 37°C. Recognition of apoptotic cells by these NRK cells was quantified by flow cytometry. (C) These NRK cells were cultured with CFSE-labeled viable cells or apoptotic cells for 30 minutes at 37°C. Percentage of the recognition was quantified by flow cytometry. Data are represented as mean ± SD of triplicates. (D) These NRK cells were pretreated with 20 μg/mL control rIgG2a, RMT3-23, or RMT4-54 mAb, and then cultured with CFSE-labeled apoptotic cells for 30 minutes at 37°C. Percentage of the recognition was quantified by flow cytometry. Data are represented as mean ± SD of triplicates. Similar results were obtained in 3 (A-C) or 2 (D) independent experiments.