Extended Data Fig. 6: Quantification of uncaging efficiency.

(a) Flow cytometric analysis of αCD8^D−1-PsT-labeled CD8⁺ T cells upon exposure to the indicated laser intensities or 1 hour to natural light. (b) Viability of uncaged (AF594^low αFLAG^high) or caged (AF594^high αFLAG^low) CD8⁺ T cells from samples exposed to the indicated laser intensities, as measured by flow cytometric analysis following IR-dye live-dead staining. Data shown are obtained from a single experiment. (c) Correlation between uncaged surface area and the observed fraction uncaged αCD8^D−1-PsT labeled CD8⁺ T cells in peripheral blood mononuclear cells (PBMCs). 0%, 50%, or 100% of the surface area of wells containing αCD8^D−1-PsT labeled cells was uncaged, and samples were analyzed by flow cytometry. (d) Quantification of the correlation between uncaged surface area and fraction uncaged CD8⁺ T cells as measured with flow cytometry, as shown in Extended data Fig. 6c. After uncaging of the indicated surface areas, cells were mixed at a 1:1 ratio with PBMCs from non-exposed samples. Cells from exposed and non-exposed samples were distinguished by labeling samples with different αCD3 antibodies before mixing. Blue line represents the fraction of uncaged (AF594^low αFLAG^high) cells after local exposure with 405-nm light. Green line represents the fraction of AF594^low αFLAG^high cells within the second sample that was not exposed to 405-nm (that is not uncaged). Note that αCD8^D−1-PsT binding is stable throughout the experimental pipeline, as demonstrated by the absence of AF594^low αFLAG^high cells in the non-exposed sample. Line graphs show mean of technical triplicates from one experiment. Data are presented as mean values ± s.d.

Source data