FIGURE 3.

Residual magnetic properties disturb purities in repetitive isolation rounds. A, Representative (of n = 3) dotplots of viable cells are shown for allo‐reactive T cell responses generated from positively selected memory T cells or untouched memory T cells (the quadrants were set based on the unstained samples). The gating of lymphocytes was based on the forward and sideward scatter after which they were plotted for CD3 and CD137 expression. After 2 wk in vitro culture of the two‐differential isolated memory T cell populations, the cells were restimulated with HLA‐mismatched EBV‐LCL. CD137 (activation marker) staining was performed 24 h after the restimulation. To isolate the allo‐reactive (activated) T cells, staining with CD137‐APC and then indirect isolation with anti‐APC magnetic beads was performed. After application on the separation columns, both the column‐retained fractions and the flow‐through fractions were analyzed for CD137 expression to assess the purity of the allo‐reactive (activated/CD137^pos) T cell isolation. B, Scatter plot with the quantification of three independent experiments shows the percentages of CD137^pos cells (black circles) and CD137^neg cells (grey squares) of total for both the CD137 negative fraction and the CD137 positive fraction. The experiment was repeated three times using three different donors. The statistical analysis was performed with t test in Prism 8 (P‐value: *<.001 and **<.00005)