Figure 4: Sequential staining of surface antigens upon fixative and permeabilization treatments.

1X106 PBMC were either surface stained (column I), surface stained then fixed in 1% paraformaldehyde (column II), surface stained, fixed in 1% paraformaldehyde, then permeabilized by 0.2% saponin (column III), fixed, permeabilized (0.2% saponin), then surface stained (column IV), or fixed, permeabilized (methanol), then surface stained (column V). Cells were stained with CD62L-FITC (clone DREG 56), CD4-PE (clone RPA-T4), CD8-PercpCy5.5 (clone SK1), and CD3-APC (clone UCHT1). Top row displays forward (FSC) and side scatter (SSC) profiles, and lymphocyte gate used for display of subsequent rows. Figure displays consequences of antigen staining towards fixation and permeabilization conditions for intracellular staining. Post permeabilization, oftentimes, reduction in the forward scatter is observed, and the appearance of these populations is illustrated by the intermediate populations appearing in the surface marker staining that are typically excluded by lymphocyte gating alone. Column VI displays the ungated surface stains alone.