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. 1999 Apr;73(4):3273–3283. doi: 10.1128/jvi.73.4.3273-3283.1999

FIG. 6.

FIG. 6

CD4-positive T cells from latently infected C57BL/6 mice do not harbor the γHV68 genome. PECs collected from C57BL/6 mice 13 to 15 days postinfection with γHV68 were stained with F4/80 or with a CD4-specific antibody for FACS sorting. The results shown represent two separate experiments. (A) Cells were pregated into lymphocyte-enriched or macrophage-enriched populations based on forward scatter and side scatter characteristics, as shown for a representative experiment. (B) PECs from the lymphocyte-enriched population were sorted into CD4-negative and CD4-positive fractions as described in Materials and Methods. Shown are the results of CD4 staining and the gates used for FACS sorting of CD4-positive and CD4-negative cell populations from a representative experiment. Gates for sorting were drawn tightly to prevent contamination of sorted populations. By these criteria, 62 to 67% of the cells from the lymphocyte-enriched gate were sorted as CD4 negative and 28% of the cells from the lymphocyte-enriched gate were sorted as CD4 positive. (C) F4/80-positive PECs were sorted from the macrophage-enriched population. Shown are the results of F4/80 staining and the gate used for FACS sorting of F4/80-positive cells from a representative experiment. For the two experiments performed, 75 to 85% of the PECs were sorted as F4/80 positive. (D) Pre- and postsorting differential analysis of Wright’s-stained cells. Cells were categorized by morphological criteria as macrophages, lymphocytes, or monocytes-lymphoblasts (Mono/Blast). Based on morphological criteria, monocytes could not always be distinguished from lymphoblasts. (E) Limiting-dilution PCR analysis to quantitate the frequency of γHV68 genome-positive cells in total PECs and F4/80-positive, CD4-positive, and CD4-negative PECs. Tenfold dilutions of each cell population were tested for the presence of the γHV68 genome by nested PCR. The data in panels D and E are averages of two separate experiments. The error bars represent the standard error of the mean. Rxns, reactions.