Gating strategy used for the FCC assay. Identification of target cells: ungated events are shown in the third panel from the left. Target cells (K562) are identified as CTO⁺ events with high side scatter. The rightmost panel, gated on target cells only, shows forward scatter versus 7-AAD fluorescence. Dead K562 target cells (color-evented purple) in A or dead Daudi cells in B are identified by 7-AAD dye uptake and low forward scatter. Identification of effector cells: the second panel from the left is gated to exclude target cells and shows the light scatter characteristics of the peripheral blood mononuclear cells assayed for cytotoxicity. The leftmost panel, gated to include lymphoid light scatter events from the previous panel, and to exclude target cells, shows anti-CD3 ECD fluorescence, versus anti-CD16 plus CD56 FITC fluorescence. Effector cells are analyzed for expression of CD3, CD16 and CD56 to identify NK (CD3⁻CD16⁺CD56⁺), NKT (CD3⁺CD16⁺CD56⁺) and T cell (CD⁺CD16⁻CD56⁻) populations.

Gating strategies used for the assessment of absolute lymphocyte and tumor cell counts in the presence of fluorobeads. Beads (high scatter events) are dark blue; live targets are orange; and dead targets are purple. A. In an open gate, fluorobeads are visualized and bead events are counted. B. Gate is set on cells, discriminating CTO + targets from lymphocytes. C. Gate is set on targets to obtain the number of live and dead K562 cells. The lower row shows the gates used to determine numbers of live an dead effector cells (NK, T and NKT).

Intra-assay variability of FCC assays performed with K562 targets and PBMC obtained from three representative normal donors (of 9 tested). Note that the Y-axis is plotted on the logit scale used to linearize the data for statistical analysis. Each FCC assay was repeated three times (solid, dashed and dotted lines).

Comparisons of the 3h FCC assay with 4 h CRA. PBMC were co-incubated with K562 targets. Non-transformed data obtained with PBMC of normal donors (n = 11) and patients with cancer (n = 15). PBMC of 12 normal donors were also stimulated with IL-2 and tested for LAK activity with Daudi targets.

Gating strategy use to determine the phenotype of effector cells (left) in FCC assays. NK cells and T cells are identified by their surface markers. On the right, NK cells and T cells are identified as CD69⁺ or Granzyme B⁺ cells in a sample of PBMC tested at 50:1 E:T ratio. NK cells show early activation and increase of the Granzyme B level, while CD69 expression and Granzyme B levels remain low in T cells after 3h incubation. This phenotype suggests that NK cells are the active cells in this assay.

Amnis images of NK cells and T cells interacting with K562 targets in the FCC assay. In A, PBMC of a normal donor are tested in the FCC assay at the E to T ratio of 6:1. In the upper left panel targets and effectors are identified on the basis of CTO staining. The targets are scored for 7-AAD uptake (killing) in the top right histogram. The brightfield aspect ratio in live (orange histogram) and dead (yellow histogram) targets reveal the presence of a high proportion of cell doublets and clusters among the dead targets. The aspect ratio measures the ratio of the width to the length of imaged objects. An aspect ratio close to 1 indicates a round object. Smaller aspect ratios indicate an elongated object, such as is formed by a cluster of two or more cells. In the bottom left histogram, a lymphoid population is defined among the CTO negative cells. There are further subdivided into NK cells (CD3- CD16+56+) and T cells (CD3+) in the bottom center histogram. In the upper right panel, the bright field image of live target cells; in the lower right panel, a bright field image of dead targets, showing the presence of a high proportion of cell doublets or clusters among the dead targets. In B, individual images of 7-AAD+ (killed) targets from the same assay (left) and binding of the NK cell or T cell to K562 targets (right). In C, individual images of live (7-AAD negative) targets with an enlarged image of the NK cell interacting with a live target. In D, individual images of 7-AAD+ targets interacting with effector cells at the 50:1 ratio. An enlarged image of a cell cluster shows the presence of NK cells and T cell conjugated a dying target.

Measurement of spontaneous release of the dye from labeled K562 cells. A representative experiment of 9 performed with PBMC of normal donors.

FCC assays performed for different co-incubation time periods using PBMC as effectors and K562 cells as targets. Representative data obtained with PBMC from one of 11 normal donors tested.

Biologic variability in NK cell cytotoxicity detected in a FCC assay with K562 cells. PBMC were obtained from the same normal donor on days 1, 30 and 99. On days 30 and 99, the FCC assays were done in triplicate. Data from a representative donor of 4 tested.

Comparisons of slopes and intercepts obtained from analyses of data obtained in normal donors (n = 11) and patients with cancer were tested with K562 targets. PBMC of 12 normal donors were stimulated in the presence of IL-2 and tested for LAK activity with Daudi targets. A line of equality (slope = 1) is drawn on each graph for reference. In all three groups, the individual intercepts are greater for the FCC assay than CRA.

Gating strategy used to evaluate conjugate formation between tumor targets and effector cells. In A, single cells (not doublets) are discriminated from cell doublets on the basis of forward scatter integral versus forward scatter peak height. Cell doublets have relatively smaller peak height for a given peak integral. Among doublets, those with highest integral forward scatter contain K562 target cells. In this reaction at the E:T ratio of 50:1 only 1.43% cells form conjugates. In B, doublets in the gate are discriminated into those that contain targets and those that do not. In B1, gate is set on targets in the doublets to determine percentages of doublets containing NK cells or T cells. Tumor-NK conjugates are the most numerous. In B2, gate is also set on targets in the doublets to determine percentages of dead (7-AAD+) targets. Nearly all conjugated targets are alive (i.e., 7-AAD-negative) after 3h of co-incubation. In C, gate is set on cells not in doublets to distinguish uncomplexed targets. In D, gate is set to determine percentages of dead (7-AAD+) targets not in doublets.