Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs by Arun T. Kamath, Sandrine Henri, Frank Battye, David F. Tough, and Ken Shortman Blood Volume 100(5):1734-1741 September 1, 2002 ©2002 by American Society of Hematology

Segregation of BrdU-labeled and unlabeled DCs Segregation of BrdU-labeled and unlabeled DCs.DCs were isolated, stained for DC surface markers and intracellular BrdU, and analyzed by flow cytometry gating on the CD11c+ MHC class IIhi population. Segregation of BrdU-labeled and unlabeled DCs.DCs were isolated, stained for DC surface markers and intracellular BrdU, and analyzed by flow cytometry gating on the CD11c+ MHC class IIhi population. Results are typical of those after 2 days of continuous BrdU administration for spleen DCs and 10 days for LN DCs. The continuous line indicates the BrdU-fluorescence staining after BrdU administration, and the broken line indicates the background staining of DCs from mice not administered BrdU but with identical, side-by-side, isolation and staining protocol. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology

BrdU-labeling kinetics of the total DC population of different lymphoid organs.BrdU was administered continually to groups of 4 to 6 mice, and the DCs were isolated at different times from the different pooled lymphoid organs. BrdU-labeling kinetics of the total DC population of different lymphoid organs.BrdU was administered continually to groups of 4 to 6 mice, and the DCs were isolated at different times from the different pooled lymphoid organs. DCs were surface stained for DC markers, then permeabilized and stained for BrdU incorporated into DNA. DC suspension was then analyzed by flow cytometry, gating for CD11c+MHC class IIhi DCs. Percentages of DCs labeled with BrdU were then determined from profiles similar to those in Figure 1. A parallel group of control mice without BrdU administration provided the DCs for the background control. Results represent pooled data from 3 to 5 separate kinetic experiments. The point near zero time was a 2-hour pulse of BrdU, which should label any DC in cell cycle; later points represent the accumulation of labeled cells from dividing precursors. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology

BrdU-labeling kinetics of subtypes of thymic DCs BrdU-labeling kinetics of subtypes of thymic DCs.Conditions were similar to those for Figure 2, except that the thymic DCs were subdivided based on surface marker differences. BrdU-labeling kinetics of subtypes of thymic DCs.Conditions were similar to those for Figure 2, except that the thymic DCs were subdivided based on surface marker differences. Upper graph: the 60% staining brightest with anti-CD8α (CD8α+) versus the 30% showing the lowest staining (CD8α−). Lower graph: the 50% showing brightest staining with anti-MHC class II(hi) versus the 50% showing less bright staining (lo). In MHC class II staining, the distribution was continuous and the division arbitrary; other cutoff points between low and high MHC class II expression gave similar results. Results are pooled from 4 separate kinetic-labeling experiments. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology

BrdU-labeling kinetics of subtypes of DCs within different peripheral lymphoid organs.Conditions were similar to those for Figure 2, except that the DCs were subdivided into discrete subtypes as detailed in the text. BrdU-labeling kinetics of subtypes of DCs within different peripheral lymphoid organs.Conditions were similar to those for Figure 2, except that the DCs were subdivided into discrete subtypes as detailed in the text. Symbols coding the subtypes are the same for all graphs. Results are pooled from 3 (mesenteric LNs) to 5 (spleen and cutaneous LNs) separate kinetic experiments. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology

Comparison of the BrdU-labeling kinetics of potential skin emigrant DCs with the corresponding DC subtypes in skin-draining LN.At various times after the administration of BrdU to mice, the incorporation of label into cutaneous LN DCs was determined as in F... Comparison of the BrdU-labeling kinetics of potential skin emigrant DCs with the corresponding DC subtypes in skin-draining LN.At various times after the administration of BrdU to mice, the incorporation of label into cutaneous LN DCs was determined as in Figure 4. At similar times (and usually from the same mice), the ear skin was removed, then cultured in a medium containing 6Ckine. DCs exiting over a 2-day period were collected and separated into the 2 subtypes, and the incorporation of BrdU was determined. Results for the skin emigrant DCs represent pooled data from 3 separate kinetic experiments. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology

Tracking the movement of DCs from skin to LN by labeling the skin with a fluorescent dye.The ear skin was painted with TRITC to mark DCs and DC precursors. Tracking the movement of DCs from skin to LN by labeling the skin with a fluorescent dye.The ear skin was painted with TRITC to mark DCs and DC precursors. Identification of skin-derived TRITC-positive gated DCs in the draining auricular LN is shown (A). DC recovery from the auricular LN of control mice before ear painting was 15 × 104 DCs per mouse, a value lower than but not statistically significant different from the recovery from ear-painted mice. The expression of DEC-205 and Langerin by the gated TRITC-positive DC is shown (B); the broken line gives the background, omitting only the specific mAb. Data represent one of 3 experiments giving similar results. Arun T. Kamath et al. Blood 2002;100:1734-1741 ©2002 by American Society of Hematology