Lymphocytes back-gating

Fig. I. Analysis of lysed whole blood stained with FITC-anti-CD45 and PE-anti-CD14. (A) Scatter analysis, showing the position of a gate to restrict the analysis to lymphocytes. (B) Gated fluorescence analysis, showing that over 98% of the gated cells are in quadrant 4 (Q4). Monocytes would appear in quadrant 2 (Q2) and nonleukocytes in quadrant 3 (Q3). With suboptimal cell preparations, the position of the lymphocyte scatter gate can be determined by back-gating that is, examining the scatter distribution of cells gated by quadrant 4.

During subsequent data analysis, gate the cells positive for CD45 and negative for CD 14 onto the FSC vs SSC dot plot to refine the optimal position for the lymphocyte scatter gate. This method is known as back-gating ... 

Fig. 6.12. Back-gating from CD14/CD45 fluorescence to determine the scatter characteristics of lymphocytes. Such back-gating facilitates the placing and then evaluation of a lymphocyte scatter gate within a peripheral blood mononuclear cell preparation.

Fig. 1. Orthogonal light scatter (side scatter, SSC) (y-axis) displayed against forward scatter (FSC) (x-axis) for the author s peripheral blood leucocytes. This is in the form of a dot plot. Each dot represents one cell. A gate has been set on the lymphocyte cluster. If there is any doubt, this cluster can be positively identified using a pan-T antibody (anti-CD3) and back-gating, as described in Section 3.2.2. Data were recorded using a Becton-Dickinson FACSCAN.

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