Migration of UCB CD34⁺ cells and Ba/F3 cells expressing human wild-type Flt3 (Ba/F3-Flt3 cells) to FL. (A) Freshly isolated CD34⁺ cells were suspended in 0.5% BSA/RPMI, and migration toward 10 ng/mL rhFL was quantitated by flow cytometry. In replicate cultures, cells were incubated with or without 10 ng/mL FL in 0.5% BSA/RPMI, and cell viability was determined based on forward and side scatter. Percentage migration of CD34⁺ cells to a positive (0/+FL; ▪) and a zero (+FL/+FL; ▴) gradient along with background migration (0/0; ○) are shown and were calculated based upon migration of CD34⁺ cells divided by fold change in viable cells incubated with FL. Data are expressed as mean ± SEM of 3 experiments. *P < .05 compared with background migration in the absence of FL. (B) Ba/F3-Flt3 cells were washed and resuspended in RPMI with 0.5% BSA at 5 × 10⁶/mL and 0.1 mL, added to the transwell with either escalating doses of rhFL in the lower chamber (0/+; •) or in both the upper and lower chambers (+/+; ○), and incubated for 4 hours at 37°C. Cells that completely migrated to the bottom chamber were enumerated, and percentage migration was calculated as described in “Materials and methods.” Data represent 1 of the 3 experiments with similar results. *P < .05 compared with background migration in the absence of FL. Error bars indicate mean ± SEM. (C) (Left) Percentage migration of control vector–transduced Ba/F3 cells (□) or Ba/F3-Flt3 cells (▪) in response to a positive gradient of increasing concentration of rhFL. (Right) Flt3 expression was analyzed by flow cytometry. *P < .05 compared with background migration in the absence of FL. Shaded curve indicates isotope staining; open curve, FIT3 staining. Error bars indicate mean ± SEM. (D) Ba/F3-Flt3 cells were extensively washed and incubated with 10 ng/mL rhFL or 100 ng/mL rhCXCL12 for up to 360 seconds, fixed, and stained as described in “Materials and methods.” The percentage increase in mean channel fluorescence of phalloidin-FITC compared with cells incubated without cytokines (•) is shown. □ indicates CXCL12; ▿, FL. Data represent 1 of the 2 experiments with equivalent results. *P < .05 compared with cells without cytokines. Error bars indicate mean ± SEM.