Fig. 2. CD4⁺ T cells are significantly and time-dependently activated in the brain after ICH.

CD4⁺ T cells were fluorescence-activated cell sorter (FACS) sorted from C57BL/6 mice and retrovirally transduced with TN-XXL plasmid. A total of 1 × 10⁷ TN-XXL–expressing CD4⁺ T cells were then injected into Rag2^−/− mice 24 hours before ICH. Twenty-four hours after ICH, FRET of TN-XXL–expressing CD4⁺ T cells in the brain, spleen, CLNs, and blood was detected using flow cytometry. (A) Schematic representation of the calcium indicator TN-XXL, containing donor fluorophore CFP, cpCitrine acceptor, and calcium-sensitive domain TnC, before and after binding of calcium. (B) After binding to free calcium, TnC undergoes a conformational change that leads to energy transfer from the donor to the acceptor fluorophore, resulting in a drop in CFP fluorescence and an increase in cpCitrine fluorescence with CD4⁺ T cells activation after ICH injury. Flow cytometry gating strategy showing negative control (mock transduced), sham operation, and ICH surgery groups in the spleen and brain separately. Bar graphs shows the FACS-based FRET measurements in TN-XXL–expressing CD4⁺ T cells within the spleen and brain at 24 hours after ICH. n = 15 per group. Two-tailed unpaired Student’s t test. (C and D) Flow cytometry histogram and measurements of FRET level in TN-XXL–expressing CD4⁺ T cells within indicated organs from 0 to 72 hours after ICH. n = 15 per group. Two-way ANOVA followed by Tukey post hoc test. Mean ± SD. *P < 0.05 and **P < 0.01.