For HPCs, CD44/β1 integrin mediates adhesion and RHAMM/β1 integrin mediates motility. (A) Sorted CD34⁺45^loSSC^lo cells were deposited into chambers coated with fibronectin, together with soluble HA, and their behavior was monitored by time lapse microscopy. Cell adhesion was defined as the number of cells remaining stationary (not floating) throughout the period of analysis. Most were deforming cells. Cell motility was defined as the number of cells that migrated a distance of at least one cell diameter during the period of analysis (13 mobilized blood and 2 steady-state BM). The values for steady-state BM were 23% to 28% motile and 36% to 61% adherent HPCs. For each sample of sorted HPCs, we analyzed the cells in at least three fields (100 to 150 total cells) to permit analysis of at least 60 adherent cells and at least 20 motile cells. The data points represent the aggregate values of all fields for each individual HPC sample. (B) For this representative sample, HPCs were pretreated with either IgG1 isotype-matched control, anti-RHAMM, anti-β1 integrin, or anti-CD44 before the time lapse microscopy and image collection. RHAMM and CD44 MoAbs serve as reciprocal internal specificity controls for inhibition of adhesion () and motility (▪), respectively. Similar experimental results for each MoAb were obtained for 3 to 6 individual HPC populations (8 mobilized blood and 1 steady-state BM). All experiments included an aliquot of sorted cells treated with an isotype-matched control MoAb and 2 to 3 of the test MoAbs. For 2 mobilized blood samples, sufficient cells were available to test the isotype control and all 3 test MoAbs in the same experiment.