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. Author manuscript; available in PMC: 2008 Aug 31.

Published in final edited form as: J Immunol Methods. 2007 Jun 28;325(1-2):51–66. doi: 10.1016/j.jim.2007.05.013

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.

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.