Phenotype of PBPCs enriched by immunoaffinity removal of lineage-postitive cells, evaluated before and after drug exposure by means of two color staining with FITC- or PE-labeled anti-CD34 antibodies (vertical axis) and PE-labeled anti-CD38 or FITC-labeled anti–P-glycoprotein antibodies, Rh 123, or Ho 33342 (horizontal axis). A total of 18 × 10⁶ PBPCs enriched as described in the Materials and Methods section were resuspended at 2 × 10⁶/mL in IMDM supplemented on day 0 by 100 ng/mL SCF, 100 ng/mL IL-3, 0.6 mg/mL 5-FU and 10% FBS. Cultures were supplemented daily by 5-FU. On day 7, cells were washed, and viability was evaluated by flow cytometry and 7AAD. About 0.7 × 10⁶ cells were still viable after drug exposure (ie, 4% of seeded cells). A total of 5,000 events were collected for each flow cytometry analysis, and the cell populations analyzed were included in the gates showed in the FSC/SSC panels. The fluorescence detectors were set up to have similar gates in all stains. Dead cells were excluded by their intense staining with 7AAD, and the percentage of positive cells per each quadrant indicated. The flow cytometry analysis shows that before drug selection (top panels) 51% to 64% of PBPCs were CD34⁺, CD38⁺, MDR⁻, Rh123 bright, and Ho33342 bright. After drug exposure (bottom panels), 88% to 94% of the cells were CD34⁺,CD38⁻, MDR⁺, Rh123 low, and Ho33342 low. Interestingly, after drug exposure the CD38⁻, MDR⁺, Rh123 low, and Ho33342 low phenotype was present also in a small (2% to 3%) but constantly present subset of CD34⁻ cells. Differences between PBPCs exposed to both mafosfamide and taxol or cultured for 7 days in the presence of 5-FU, SCF, and IL-3 were not significant. The results of one representative experiment of 7 are shown.