Journal: eLife

Article Title: Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice

doi: 10.7554/eLife.96394

Figure Lengend Snippet: ( A–B ) Comparison of MHCII MFI in YFP + and YFP - pDCs from Cd79a -Cre-YFP reporter mice via flow cytometric analysis. ( C–D ) Cd79a -Cre-YFP mice were injected with 50 µg/mL (100 µL) of CpG:ODN or control GpC:ODN and analyzed via flow cytometry for splenic pDC numbers/100,000 cells. ( E ) Flow cytometry quantification of YFP - pDC and B-pDCs (YFP + ) fold change in cell numbers upon CpG:ODN in vivo challenge. ( F–G ) Fold change in CD86 + CD89 + cell numbers for each pDC population in mice restimulated with CpG:ODN. Difference in CD86 + CD89 + cell numbers between stimulated B-pDCs and YFP - pDCs was also significant. ( H ) In vitro Toll-like receptor 9 (TLR9) engagement of B-pDC or pDC for ELISA against IFN-α orIL-12p40. ( I ) T cells were magnetically isolated from wildtype C57BL/6J mouse splenocytes using MACs columns, labeled with CSFE, and then cultured (2.5×10 4 /well) alone or with CpG:ODN activated B-pDCs or pDCs (5×10 3 /well) for 6 days. ( J ) The percentage of CFSE-negative CD3 + T cells in co-cultures were significantly higher in B-pDC compared to pDCs. Mann-Whitney t-tests were used for all statistical comparisons. Error bars = mean ± s.d. The results are representative of three independent experiments each containing at least 4 mice per group.

Article Snippet: Preactivated pDCs (5×10 3 ) were cultured with lymphocytes (2.5×10 4 ) magnetically sorted from mouse spleen single-cell preparations using MACs columns and the EasySep Mouse T Cell Isolation Kit (StemCell Technologies Cat# 19851) for a total of 6 d with 20 units/mL of IL-2.

Techniques: Comparison, Injection, Control, Flow Cytometry, In Vivo, In Vitro, Enzyme-linked Immunosorbent Assay, Isolation, Labeling, Cell Culture, MANN-WHITNEY