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Figure 6 | BMC Immunology

Figure 6

From: Performance of plate-based cytokine flow cytometry with automated data analysis

Figure 6

Automated gating of CFC samples. (A) Example of automated gating. A cluster-finding algorithm is employed to automatically identify small lymphocytes in forward versus side scatter (R1, top left panel; this region can also be drawn manually during sample acquisition). A similar automated region is calculated for CD3+CD8- and CD3+CD8+ lymphocytes (R2 and R3, top right panel). R2 and R3 are set for maximal size to allow inclusion of activated cells that have down-modulated CD3 and CD8 (bold dots). The lower left plot, gated on regions R1 and R2 (CD3+CD8- lymphocytes), uses the cluster-finding algorithm to identify the CD69-IFNγ- population (R4). This population is tethered to a rectangular region that identifies the CD69+IFNγ+ cells (R5). The lower left plot is similar, but gated on R1 and R3 (CD3+CD8+ lymphocytes). The percentage of gated events in R5 is reported. (B) Correlation of the automated gating template to expert manual gating. Whole blood from 23 CMV seropositive donors was stimulated in deep-well 96-well plates, then analyzed manually or using the automated template in (A). Using a batch analysis protocol, many CFC samples can be analyzed and the resulting data downloaded to a spreadsheet in a rapid fashion.

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