In vitro and in vivo ASC survival is dependent on CD138 expression. (A-D) Purified B cells from WT and sdc1-deficient mice (sdc1^−/−) were cocultured in vitro with LPS for 3 days and ASC generation (YFP⁺) was examined. (A) Purified B cells from WT and sdc1^−/− mice were pre-labeled with celltrace violet, and analyzed for cell division up to day 6 of culture. Resting B cells cultured in the absence of LPS were used as a control. (B) Flow cytometry gating strategy depicting a contour plot of YFP⁺ ASCs generated following culture. WT (blue) ASCs were distinguished from CD138-deficient ASCs (red) by the expression of CD45.1 using flow cytometry. (C) The percentage of YFP⁺ ASCs generated by in vitro culture is demonstrated on day 3 and day 6 and expressed as a percentage of total cells. (D) YFP⁺ ASCs derived from WT B cells were examined for apoptosis on day 3 after culture with LPS. Mature CD138⁺YFP⁺ ASCs were distinguished from immature CD138⁻ ASCs by flow cytometry, and apoptosis was examined using Annexin V or activated caspase 3 staining. (E) Human PBMCs were examined by flow cytometry. Gating on CD19^lowCD20⁻CD38^high cells revealed CD138⁺ and CD138⁻ fractions that were examined for Annexin V staining. Representative contour plots are demonstrated. (F-K) YFP⁺ ASCs were generated in vivo following co-transfer of purified naïve B cells from WT and sdc1^−/− mice and immunization with NP-OVA (protocol depicted in Figure 2A). (F) Cells from the dLN of immunized mice were stained for Annexin V to examine apoptosis and analyzed by flow cytometry. The percentage of apoptosis in YFP⁺ ASCs or YFP⁻ transferred cells is depicted (WT [blue]; sdc1^−/− [red]). (G) Within the WT ASC compartment, mature CD138⁺YFP⁺ ASCs were distinguished from immature CD138⁻ ASCs by flow cytometry. A representative dot plot is shown (left). The graph represents the percentage of cell apoptosis within the CD138⁺ and CD138⁻ subsets in dLN of immunized mice (right). (H) Intravital two-photon microscopy was performed on the surgically exposed popliteal LNs of immunized mice. Myeloid cells (blue), ASCs (green), naive B cells (red), and collagen (purple) could be distinguished from one another. A representative image of an apoptotic event is depicted. Apoptotic events were recorded and quantified in a blinded manner (left). Graph represents the number of apoptotic events observed per hour (right). (I-J) Cells from the dLN of immunized mice were stained for Bcl-2 (I) or Mcl-1 (J), and analyzed by flow cytometry. Graph depicts MFI in YFP⁺ ASCs from either WT (blue) or sdc1^−/−-derived (red) cells. Within the WT ASC compartment, mature CD138⁺YFP⁺ ASCs (open circles) were distinguished from immature CD138⁻ ASCs (closed circles) by flow cytometry, and graph depicts MFI. FMO controls included. (K) dLN cells were stained with ki67 to examine proliferation by flow cytometry. WT YFP⁺ (red) and CD138-deficient YFP⁺ (blue) ASCs are depicted in a representative histogram. *P < .05; **P < .01; ***P < .001. dLN, draining lymph nodes; FMO, fluorescence minus one; ns, not significant; PBMCs, peripheral blood mononuclear cells.