Zn accumulates in thymocytes and is released after damage. (A-B) Six- to 8-week-old female C57BL/6 mice were given 550 cGy TBI, and levels of Zn were measured by ICP-MS. (A) Total thymic amounts of Zn from both intracellular and extracellular fractions of thymus (n = 6/timepoint). (B) Extracellular Zn was measured only in thymic supernatants, and the ratio of extracellular to total thymic Zn was calculated (n = 6/timepoint). (C) Six- to 8-week-old female C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO₄) for 21 days, at which point 1 cohort was given 550 cGy of TBI. Thymocytes were isolated either before or 48 hours after TBI and cocultured with exECs. Bmp4 expression was measured by qPCR at 24 hours (n = 3-4/group). (D) Six- to 8-week-old female C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO₄) for either 21 days before TBI or from the day of TBI and maintained on ZnSO₄ in drinking water for the duration of the study. Thymus cellularity was measured at day 28 after TBI (n = 4-5/group). (E) Six- to 8-week-old female C57BL/6 mice were fed a normal or ZD diet for 21 days, after which thymocytes were isolated by CD90⁺ magnetic separation. Intracellular Zn levels were measured by staining with Fluozin-3 and assessed by flow cytometry (n = 5/group across 2 independent experiments). (F-G) Six- to 8-week-old female C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO₄) for 21 days, after which thymocytes were isolated by CD90⁺ magnetic separation and Zn was measured by staining with Fluozin-3 (F) or ICP-MS (G). Graphs represent mean ± SEM; each dot represents a biologically independent observation. *P < .05; **P < .01; ***P < .001.