Using a live/dead to remove dead cells can improve your staining. (a). Use of forward and side scatter gating (red rectangle) may not remove all dead cells and some non-specific binding may still be present. (b). Exclusion of dead cells using propidium iodide staining (red rectangle) means less non-specific binding and easier identification of positively stained populations. Images shown here are human peripheral blood stained with CD14 (MCA1568) and CD3 (MCA463).
Sequential gating to identify specific T subsets. Red cell lysed whole blood was stained with CD3 (MCA463P750), CD4 (MCA1267PB), CD8 (MCA1226PE), CD28 (MCA709A647) and CD45RA (MCA88F) in the presence of propidium iodide. The gating strategy is shown by the red arrows.
Backgating to identify leukocyte subsets. (a) Red cell lysed whole blood was stained with CD3 (MCA463A647) and CD14 (MCA1568P750) (b). Cells in gates A, B and C were backgated onto FSC vs SSC (c) to identify specific leukocyte populations.
Figure A. FITC conjugated Rat anti Human CD18 (MCA503F) and RPE conjugated Mouse IgG1 isotype control (MCA928PE). Figure B. FITC conjugated Rat anti Human CD18 (MCA503F) and RPE conjugated Mouse anti Human CD19 (MCA1940PE).