Neutrophils migrate from the ileum to the mesenteric lymph node. Mice transgenic for the photoconvertible green-to-red protein Dendra2 in all cells underwent TBI. After 10 or 34 hours, the ileum was exposed to 405-nm light during surgery to convert Dendra2 from its green to the red fluorescent form. Mice were sacrificed 10-14 hours after illumination and the amount of photoconverted cells that had trafficked from ileum to spleen, mLN, ndLN (nondraining lymph node, pooled iLN and aLN), blood, and BM was determined. (A) Illustration of experimental setup. (B) Representative fluorescence image showing successful photoconversion selectively in the ileum after surgery. (C) Representative FACS plots of photoconverted CD45⁺CD11b⁺Ly6G⁺ cells 48 hours after TBI in different organs. Pixel size of dots was increased for better visibility. (D-E) Percentage of photoconverted CD11b⁺Ly6G⁺ neutrophils is shown. (D) Photoconversion on day 0, 10 hours post-TBI, and analysis on day 1, 24 hours post-TBI, according to setup A1 (n = 6 for each organ from 2 independent experiments). (E) Photoconversion on day 1, 34 hours post-TBI, and analysis on day 2, 48 hours after TBI according to setup A2 (n = 7 for each organ from 2 independent experiments). (F-G) Neutrophils from the BM of mice transgenic for the photoconvertible green-to-red fluorescence protein Dendra2 were isolated and enriched by MACS purification. The cells were photoconverted ex vivo, and 10⁷ neutrophils were adoptively transferred into C57BL/6 mice 34 hours after they underwent TBI. Fourteen hours after injection and 48 hours after TBI, mice were sacrificed, and photoconverted Dendra2 cells in spleen, mLN, ileum, and blood was analyzed by flow cytometry. (F) Representative FACS plots of photoconverted CD45⁺CD11b⁺Ly6G⁺ cells derived from the spleen. (G) Percentage of photoconverted CD11b⁺Ly6G⁺ neutrophils in different organs is shown (n = 8).