1. Flow-Enhanced Stability of Rolling Adhesion through E-Selectin
Quhuan Li, Annica Wayman, Jiangguo Lin, Ying Fang, Cheng Zhu, Jianhua Wu 
Biophysical Journal 
Volume 111, Issue 4, Pages (August 2016)
DOI: /j.bpj
Copyright © 2016 Biophysical Society Terms and Conditions

2. Figure 1
Schematics of the invertible flow chamber and cell rolling. (A) Left: The flow chamber was assembled with two Lexan parts (length × width × height = 7.457 × 3.73 × 0.589 cm), a 0.01-cm-thick plastic shim gasket (cut to create a working space with 4.3 × 1.3 × 0.01 cm), a coverglass (length × width × thick = 60 × 22 × 0.16–0.19 mm), and six screws. Right: An HL-60 cell was perfused over a surface coated with E-selectin. (B) Primary capturing, rolling, detaching, and reattaching cells were observed in the upright orientation. (C) Only stably rolling cells were observed in the inverted orientation, as gravity tended to drive the cell away from the surface once the last bond dissociated, thereby limiting reattachment. A minimal model for rolling is depicted where a rolling cycle consists of a stop (1st and 3rd cells) and a go (2nd cell) period and alternates between a two-bond (1st and 3rd cells) and a one-bond (2nd cell) phase. The cell detaches if no new bond has formed during the one-bond phase before it dissociates. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

3. Figure 2
Rolling step model. The plots for probability of rolling N steps, pN versus time, t (N = 1–10) (A), and versus number of rolling steps, N (t = 5 and 10 s) (B), were calculated from Eq. 1 using parameters (koff/k = 3.30E-4, koff0 /k = 2.99E-4, kts = 1661) measured from HL-60 cells rolling on surfaces coated with 40 ng/mL E-selectin in inverted orientation at 0.5 dyn/cm2 wall shear stress. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Comparison between experimental and theoretical cell flowing velocities. (A) Representative instantaneous velocities (measured at 100 fps) of 6-μm-radius HL-60 cells flowing over and being arrested on a surface coated with E-selectin at five wall shear stresses (indicated). (B) Mean ± SD velocities of flowing cells (three experiments, ≥30 cells/point) were plotted versus wall shear stress (red triangle) and fitted to a straight line (red solid line, R2 > 0.9). Data were compared to theoretical predictions that assume a 0.1 μm (blue dashed line) or 0.4 μm (green dotted line) gap distance between the cell bottom and the surface. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

5. Figure 4
Rolling velocity and tether lifetime. (A) Velocity versus wall shear stress of HL-60 cells rolling on an E-selectin-coated (40 ng/mL) surface in normal (blue diamond) and inverted (red triangle) orientations, showing triphasic dependence. (B) Lifetime versus wall shear stress for HL-60 cells transiently tethered to E-selectin-coated (10 ng/mL) surface. Data are presented as the mean ± SD of three independent experiments. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Observation of cell rolling behavior. (A) HL-60 cells were perfused at 0.4 dyn/cm2 wall shear stress through the upright (left) or inverted (right) flow chamber whose surface was coated with 40 ng/mL E-selectin. Cell motion was tracked at 100 fps and cell trajectories over a 0.1-s period are depicted as lines connecting to the color-matched cells imaged at the first frame. Scale bar, 100 μm. (B) Quantification of (A) based on observing >60 cells in each chamber. Rolling, skipping, and detachment are defined by displacements of <10 μm, between 10 and 200 μm, and >200 μm, respectively. The only tethered cell was identified by its lack of motion over 0.05 s. The numbers of cells analyzed are indicated above the bars. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Instantaneous rolling velocity. Representative instantaneous velocity (recorded at 100 fps) versus time that individual HL-60 cells rolled in the upright orientation (left column) and inverted orientation (right column) at the indicated wall shear stress.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Rolling step analysis. Mean stop time (A), fractional stop time (B), stop frequency (C), and rolling step time (D) of HL-60 cells rolling on surfaces coated with E-selectin (40 ng/mL) in the upright and in inverted orientations are plotted against wall shear stress. Data are presented as the mean ± SD of three independent experiments. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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9. Figure 8
Detachment of rolling cells before and after lowering the flow rate. HL-60 cells were perfused over the E-selectin-coated surface (40 ng/mL) at 0.4 dyn/cm2 in the upright orientation to establish steady-state rolling before inverting the flow chamber. The wall shear stress was changed to 0.1 dyn/cm2 (left column) or 0.35 dyn/cm2 (right column) during inversion. Images were taken immediately (top row) or 20 s (bottom row) after inversion to observe detachment of HL-60 cells. Scale bar, 100 μm. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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10. Figure 9
Duration of rolling adhesion. Mean ± SD duration (≥60 measurements per point) (A) and detachment rate, koff,RA, of rollingly adherent cells (three independent experiments for each wall shear stress) (B) versus wall shear stress plots of rolling adhesion of HL-60 cells on surfaces coated with 40, 60, and 80 ng/mL E-selectin. Ln(number of cells with a rolling lifetime ≥t) (C and D) and fraction of cells that remained rolling (E and F) on 40 ng/mL E-selectin were plotted versus time and fitted to the first-order detachment model (C and D) or the model for rolling adhesion (E and F) at 0.1, 0.2, and 0.3 dyn/cm2 (C and E) or 0.5, 0.6, and 0.7 dyn/cm2 (D and F). The following procedures were used to fit the fraction of rolling cells versus time plots. For a given wall shear stress, we looked up the corresponding koff (reciprocal mean tether lifetime) from Fig. 4 B and obtained koff0 = 1.2 s−1 by extrapolating the curve to zero force (Fig. 4 B), which agrees with a previously published value (34). We then looked up ts and tc from Fig. 7, A and D, respectively. Using the tc value, we determined the maximum number of steps t/tc (nearest integer value) for each time point, t, and calculated pN for N = 1, 2, …, t/tc using the above koff and ts values plus a guess value for k, as illustrated in Fig. 2. The predicted value for PRA = p1 + p2 + … pt/tc was then calculated and compared to the experimental value to determine the error between prediction and measurement at that time point. A Matlab program using the lsqcurvefit function was employed to minimize the total error accumulated from all time points, which returned the best-fit k value for that wall shear stress. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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11. Figure 10
Kinetics parameters of rollingly adherent HL-60 cells on E-selectin. HL-60 cells rolling in the inverted orientation were detached from the ceiling coated with 40, 60, 80 ng/mL E-selectin at various wall shear stresses. The overall rate constant (A) and the apparent molecular on-rate (B) as functions of wall shear stress were derived from the model fitting of probability of rolling adhesion versus time. The effective 2D affinity (C) was calculated by dividing the effective 2D on-rate by the off-rate at each level of wall shear stress. Data are presented as the mean ± SD of three independent experiments. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions