Identifying Therapeutic Targets for Sepsis Research: A Characterization Study of the Inflammatory Players in the Cecal Ligation and Puncture Model

Figure 1

Example of a gating strategy for flow cytometry (panel 3—T cell and B cell stain and panel 4—Treg stain). Leukocytes were gated based upon their forward scatter (FSC) and side scatter (SSC) properties in order to exclude debris (a), followed by the exclusion of doublets based upon the forward scatter height (FSC-H) versus the forward scatter area (FSC-A) plot (b). Next, the percentage of CD3⁺ cells (effector T cells) was identified in the singlet cells gate gated upon the expression of CD3 and SSC properties (c). Subsequently, the differentiation was made in this population between the number of CD4⁺ cells to identify helper T cells or CD8⁺ cells to identify cytotoxic T cells (d). In the singlet gate, the percentage of CD4⁺ cells (helper T cells) was then identified (panel 2) (f) and subsequently the CD25⁺Foxp3⁺ cells were identified in the CD4⁺ gate (f) to identify regulatory T cells. In the leukocyte gate, the percentage of CD19⁺ cells was also determined to identify B cells (g). Cell subsets were expressed as a percentage of the total leukocyte count. FMO samples were implemented as appropriate gating controls. A: area; CD: cluster of differentiation; Foxp3: forkhead box p3; FSC: forward scatter; H: height; SSC: side scatter; Treg: regulatory T cells.