Migration of hematopoietic cells in response to the combination of FL and CXCL12. (A) Migration of freshly isolated UCB CD34⁺ cells to a positive gradient of escalating doses of CXCL12 was analyzed in the absence (□) or presence of 10 ng/mL rhFL (▪) in the bottom chamber for 4 hours. Data are the mean ± SEM of 2 experiments. † denotes synergistic effect of FL plus CXCL12. (B) Migration of CXCR4⁺, Flt3⁺, and CD34⁺ cells to 10 ng/mL FL and/or 100 ng/mL CXCL12. Partially purified UCB CD34⁺ cells were stained with FITC anti-CD34, PE anti-Flt3, and Cy-chrome anti-CXCR4, and CD34⁺, CXCR4⁺, and Flt3⁺ cells (gate R1) were sorted by FACS. The isotype and CXCR4/Flt3 staining are shown. Cells were subjected to migration for 4 hours. Data represent the mean ± SEM of 2 experiments. † denotes synergistic effect of FL plus CXCL12. (C) Total CD34⁺ cells were subjected to migration to 10 ng/mL rhFL and/or 100 ng/mL rhCXCL12 for 4 hours. Intracellular Ki-67 expression in CD34⁺ cells was performed as described.⁴⁰  Migrated cells were fixed with 1% paraformaldehyde overnight at 4°C and washed with PBS containing 0.25% Triton X-100 and 1% BSA. Cells were blocked with human IgG on ice for 10 minutes and then stained with FITC-conjugated isotype control or anti-Ki67 antibody in the same buffer on ice for 60 minutes. Cells that appeared below isotype staining were defined as Ki-67_negative cells. The percentage of Ki-67_negative cell migration (▪) was calculated as the number of migrated Ki-67_negative cells divided by the number of Ki-67_negative CD34⁺ input cells multiplied by 100. A similar calculation was performed for Ki-67_positive cells (□). Data are the mean ± SEM from 4 experiments. *P < .05 compared with Ki-67_positive cells by t test; † denotes synergistic effect of FL plus CXCL12. (D) Percentage migration of Ba/F3-Flt3 cells to FL, CXCL12, or the combination of FL and CXCL12. Exponentially growing Ba/F3-Flt3 cells were extensively washed and 5 × 10⁵ cells subjected to in vitro transmigration assay to 10 ng/mL rh FL, 100 ng/mL rmCXCL12 or rhCXCL12, or FL plus CXCL12. Data are shown as mean ± SEM percentage migration for 3 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12. (E) Percentage migration of Ba/F3-Flt3 cells toward FL (10 ng/mL) or CXCL12 (100 ng/mL) in the presence or absence of negative gradient of FL (10 ng/mL). Data are shown as mean ± SEM percentage migration for 3 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12. (F) Migration of the human RS4;11 cells in response to 10 ng/mL FL and/or 100 ng/mL CXCL12. Cells were extensively washed and evaluated for migration for 4 hours in 0.5% BSA/RPMI. The data represent the mean ± SEM percentage migration for 1 of 2 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12.