Fig. 2

Flow cytometric analysis of T_reg cells along the manufacturing process. Whole blood (starting material) or in-process samples (postdepletion, postenrichment, and postexpansion) were stained with fluorochrome-conjugated antibodies against surface markers CD45, CD4, CD25, and CD127 in TruCOUNT tubes and analyzed according to the lyse-no wash method. A gating strategy for T_reg cell enumeration in whole apheretic samples. A (step 6) to D T_reg cell purity quantification along the manufacturing process: starting material (A step 6), postdepletion (B), postenrichment (C), and postexpansion (D) fractions. In detail: Trucounts for absolute cell counts were identified by the intersection of events gated based on standard light scattering characteristics and those gated based on fluorescence, as shown in a PE vs. FITC plot with both plots showing all acquired events (step 1). Cell aggregates and debris were excluded on an FSC-A vs. FSC-H dot plot (step 2), followed by CD45⁺ cell identification on an APC-H7 vs. SSC-A dot plot (step 3). Among CD45⁺ cells, viable cells were gated based on negativity for 7-AAD staining (step 4). Within viable CD45⁺ cells, cells expressing CD4 or CD8 were detected in a PE vs. FITC plot (step 5), whereas CD4⁺CD25⁺ T_reg cells were identified in a FITC vs. APC plot (step 6). Finally, among T_reg cells, CD127⁻ cells were gated on a PE-Cy7 vs. APC dot plot (step 7). For samples with a higher content of T_reg cells (postenrichment and postexpansion), twin samples were stained for a surface marker in FACS tubes and then fixed and permeabilized for intracytoplasmic staining of FoxP3, which was detected among CD127⁻ T_reg cells on a PE vs. APC dot plot (step 8). Representative images of samples from patient KD2 are shown. The negative control for FoxP3 staining is shown in Additional file 1: Fig. S1