Figure 2

Gating strategy established for flow cytometric quality and in-process control regarding the CliniMACS CCS validation. Samples of the collected CliniMACS CCS fraction were analysed by flow cytometry using the Quality control panel QCP-A/A⁻ and the represented gating strategy. All cell fractions (leukapheresis, original fraction (OF), T-cell fraction (TCF), negative fraction (NF), waste fraction (WF), 48 h, 54 h, and 72 h post-leukapheresis (Stabi48, Stabi54, and Stabi72)) were stained with specific antibodies to visualize IFN-γ⁺ T cells. In the first plot, cells were analysed by 7AAD viability staining to determine the live versus dead cells, followed by gating cells based upon CD45 expression to identify CD45⁺ leukocytes in the total viable 7AAD⁻ population. In the next gating step, T cells were selected based on CD3 expression. CD3⁺CD56⁺ NKT cells were gated out using a dump channel. CD4 and CD8 surface expression was then determined from this gated population. IFN-γ⁺ T cells were gated on CD3⁺CD56⁻ T cells and on the CD4⁺ and CD8⁺ subpopulation of CD3⁺CD56⁻ T cells. The axes of the dot plots are biexponential.