The approach used in identification of NP-specific ASCs in the blood. (A) A sequence of flow cytometry plots used to identify NP-specific ASCs in the blood. eFluor780⁻TCRb⁻ cells high for the intracellular NIP and IgG1 signals show a distinct population of CD138⁺Irf4^hi cells (rightmost plot). The number of such eFluor780⁻TCRb⁻CD138⁺Irf4^hiNIP⁺IgG1⁺ cells per 1 ml blood was calculated as described in Materials and methods. (B) Flow cytometry plots showing proportions of aCasp3⁺ cells found in various splenic cell populations of the same mouse. eFluor780⁻CD4⁻CD8⁻IgD⁻CD138⁺CD267⁺ cells high for intracellular IgG1 but not for NIP were generally found to have very few aCasp3⁺ cells, presumably due to the fact that lower rates of their generation were not exceeding the rates of their elimination from the tissue. For all flow cytometry plots, the numbers next to the enclosed areas (gates) represent percentages of the cells falling into the gate, and the number in the bottom right corner shows the number of cells in the plot. Arrows denote the hierarchical relationship between the cell populations. (C) A plot showing proportions of aCasp3⁺ cells in the population of CD138⁺NIP⁺IgG1⁺ cells identified as in Fig. 1 D, Fig. 3 E, and Fig. S3 B in the spleens of Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) mice taken at day 5 following reimmunization with NP-KLH in an independent experiment. Each symbol represents data from an individual mouse, and lines represent means (n ≥ 5; the P value is for a Student’s t test). Pos, positive.