Desensitization of CXCR4. Nonexpanded CD34⁺ cells were pretreated with SDF-1 (200 ng/mL) for 1 hour, 20 hours, or kept in medium alone. Cells were washed and either immediately loaded with Fluo-3AM or kept in medium for 1 hour or 4 hours (in the absence of SDF-1) and then loaded with Fluo-3AM. The time noted in parentheses in the insert key box denotes the time for which SDF-1–pretreated cells were kept in medium prior to SDF-1 stimulation. Baseline fluorescence was first recorded and calcium flux following SDF-1 (200 ng/mL) stimulation (indicated by ↑) was analyzed by FACScan. Increase in fluorescence intensity following SDF-1 addition (compared with baseline), represents the calcium flux in cells and is shown on the y-axis of the plot. Data represent mean ± SEM of 3 independent experiments. *P < .05 and **P < .05 compared with cells pretreated in with SDF-1 for 1 hour and 20 hours, respectively.