The proportion of T lymphocytes exhibiting the CD4^lo phenotype can be found among anti-CD4–stained peripheral blood lymphocytes.

The cell suspension is stained with FITC–anti-CD3 and PE–anti-CD4 monoclonal antibodies and analyzed by FACS. Only CD3⁺lymphocytes are included in subsequent analysis. The flow cytometry histogram contains the overlay of the fluorescence intensity of the isotype control (white, control) and the fluorescence distribution for the cells stained with PE-conjugated anti-CD4 antibody (shaded). Boundaries of the CD4^lo subpopulation (darkly shaded) are found as follows: 1, a marker is set to delineate the CD4⁻population (closely overlapping the isotype control); 2, a marker containing all CD4⁺ cells (total CD4⁺) is stretched from the higher end of the first (CD4⁻) marker; 3, a position of the modal value for the CD4-PE fluorescence is found; 4 and 5, an “upper” half of the fluorescence peak containing the CD4⁺ cells is delimited, and mirrored toward the lower fluorescence values to cover all of the CD4⁺ population; 6, the CD4^lo subpopulation is assigned the position between the beginnings of the second (“total” CD4⁺) and fifth (“true” CD4⁺) markers.