Fig. 6.

LAK cell-mediated killing of K562 targets is unaffected by staining with CellVue Claret. LAK cells were labeled with CellVue Claret and incubated with PKH67-labeled K562 cells at effector to target (E:T) ratios ranging from 100:1 to 0.8:1 for 4 h at 37°C. Test samples and controls (Table 2) were analyzed using the gating strategies described in Fig. 5. (a) Representative plots from test samples with 50:1 and 3:1 E:T ratios and from the K562 target only and LAK cell only controls. (b) LAK-induced cytotoxicity of K562 cells was assessed for each condition as described in Fig. 5 using either Method 1 (based on percent of target cells that took up 7-AAD, Subheading 3.3.4, step 5; squares) or Method 2 (based on number of viable target cells remaining in the presence versus absence of effectors, Subheading 3.3.4, step 6; circles). As an internal control, percentage of dead LAK effectors (triangles) was assessed by Method 1 at each E:T ratio and verified to be acceptably low and relatively constant. To determine whether CellVue Claret staining affected their cytolytic potential, parallel studies were performed using CellVue Claret-stained (solid lines) and unstained (dashes) LAK effectors. The data indicate that LAK cells kill K562 cells in a concentration-dependent manner and that the tracking dye did not affect function. Interestingly, Method 2 was slightly more sensitive at detecting target cell loss (Note 43). Representative data from one of two replicate experiments are shown; data points signify the mean ± 1 standard deviation of triplicate samples.