Extended Data Fig. 2: Generation of stable dimeric αCD8 nanobodies.
From: Single-cell analysis of regions of interest (SCARI) using a photosensitive tag
(a) Binding stability of αCD8 nanobodies to CD8+ T cells. Top panel: a first population of CD8+ T cells was stained with FITC labeled monomeric nanobody (αCD8M-FITC) and a second population was stained with αCD8M-AF647. Bottom panel: a first population of CD8+ T cells was stained with FITC labeled dimeric nanobody containing recognition domains that are identical to the αCD8M (αCD8D−2-FITC) and a second population was stained with αCD8D−2-AF647. Subsequently, two cell populations labeled with either monomeric or dimeric αCD8 reagents were mixed, incubated at 37oC, and exchange of αCD8 nanobodies was measured by flow cytometry. Quantification of this αCD8D−2 clone and of a second nanobody clone αCD8D−1 is depicted in Extended data Fig. 2b. (b) Data depict AF647 fluorescence signal of two CD8+ T cell populations that were either stained with the indicated αCD8D-AF647 or with the corresponding αCD8D-FITC, and that were mixed and subsequently incubated at 37 °C. Note the lack of substantial AF647 signal on the FITC-labeled bystander pool. Data are representative of 2 independent experiments.
