For FRAP experiments, Ba/F3-FcγRI-eYFP cells were washed twice in PBS and seeded in a fibrin matrix: fibrinogen (2.5 mg/ml) and thrombin (1 × 10−4 U/μl) in RPMI 1640 without phenol red supplemented with 1% FCS/2 mM l-glutamin on a μ-Dish (35 mm, high; ibidi). The cells were incubated overnight in this matrix; to prevent dehydration of the matrix, RPMI 1640 without phenol red with 1% FCS/l-glutamin was added. The next day, FRAP measurements were performed on these cells (no IL-3) or after IL-3 stimulation for 30 min (IL-3). FRAP experiments were performed on a Zeiss LSM 710 confocal microscope with a 63× oil objective lens and an environmental chamber for temperature (37°C) and CO2 (5%) control. An argon laser provided the 488-nm excitation. Ten prebleach images were acquired, after which a small area (~1 μm2) spanning the membrane was bleached for 0.2 s to obtain a bleach of ~50%. The fluorescence in this region was monitored by acquiring images at 7.3 frames/s for 30 to 35 s per cell. For each condition, >70 cells were measured.

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