Characterization of viable versus apoptotic cells by a dual staining flow cytometry technique. Resting, murine B cells were cultured either without stimulant (A) or with 50 μg/mL lipopolysaccharide for 48 hours. The cells recovered at 48 hours were dually stained with Hoechst 3342 and merocyanine 540 (MC540) as described460 and the cells were analyzed on a FACS Star Plus flow cytometer (Becton Dickinson, San Jose, CA). Dual parameter dot plots enabled the identification of five distinct subpopulations defined as follows: R1, cells with 2n DNA that were MC540 negative/dull (red dots); R2, cells with greater than 2n DNA that were MC540 negative/dull (green dots); R3, cells with 2n DNA that were MC540 bright (blue dots); R4, cells with greater than 2n DNA that were MC540 bright (brown dots); and R5, cells that displayed reduced Hoechst 33342 staining and were either G₀ /G₁ (R1) or S/M/G₂ (R2) cell cycle stages. The R3 and R4 subgroups represent cells in early stages of apoptosis, whereas the R5 subgroup represents fragmenting apoptotic cells. Most techniques for evaluating percentages of apoptotic cells detect only the cells localized to the R5 subgroup. Figure courtesy of E. Charles Snow, PhD.