Extended Data Figure 5. LIPSTIC labeling ex vivo.

a, Experimental setup followed in b–c to assess the influence of substrate incubation length on intercellular labeling between primary DCs and CD4⁺ T cells. Cd40^G5/G5 DC populations were separately pulsed with 1 μM of OVA^323-339 or LCMV-GP^61-80, mixed, and co-cultured for 6 hours with Cd40lg^SrtA/Y CD4-Cre OT-II CD4⁺ T cells. Biotin-LPETG was added during the final 1, 5, 10, 20, 180 min of co-culture or for the entire co-culture time (360 min) at a final concentration of 10 μM, and cells were analyzed by flow cytometry. b, Flow cytometric analysis of co-cultured DCs incubated with biotin-LPETG for the indicated times. c, Percentage of biotin⁺ DCs gated as in b. d, Experimental set-up followed in e–g to probe intercellular labeling ex vivo between primary B cells and CD4⁺ T cells. Two populations of Cd40^G5/G5 B cells that either carried a WT polyclonal B cell receptor (BCR) repertoire or expressed the B1-8^hi Ig heavy chain, which when paired to an Igλ light chain confers specificity towards the hapten 4-Hydroxy-3-nitrophenylacetyl (NP), were mixed and pulsed with the indicated concentrations of NP-OVA. Cells were then co-cultured for eighteen hours with Cd40lg^SrtA/Y CD4-Cre OT-II CD4⁺ T cells. Biotin-LPETG was added during the last thirty min of co-culture at a final concentration of 100 μM, and cells analyzed by flow cytometry. e, Flow cytometric analysis of B cells pulsed with 1 nM of NP-OVA showing preferential biotin labeling of B1-8^hi λ⁺ B cells. f, Percentage of biotin⁺ B cells among polyclonal and B1-8^hi λ⁺ populations at the indicated NP-OVA concentrations. g, Flow cytometric analysis of B cells pulsed with 1 nM of NP-OVA showing positive correlation between biotin labeling and expression of the activation marker CD69. Data representative of three independent experiments.