## 2 Methods
### Coupled Side-View \(\upmu\)PIV System Modification and Experimental Procedure  
We have a methodology for studying the local fluid dynamics around a PMN and a melanoma cell with five PMN-to-TC position states under different flow conditions [22]. A side-view flow chamber with an inner dimension of 700 \(\upmu\)m (width) \(\times\) 550 \(\upmu\)m (height) was assembled by inserting a Vitrobes #5005 microslide into a Vitrocolls #8270 microslide (Vitrocom; Mountain Lakes, NJ, USA), which were sterilized. Two needles were then used to connect the tubing and the chamber. Finally, superglue was used to seal the chamber. As the aspect ratio was 1.27, we followed the method described by [PERSON] et al. to estimate wall shear stress distributions by applying a correction factor to analytical 2D channel velocity profiles [29]. The side-view \(\upmu\)PIV system set-up was mainly as previously described [22], and consisted of a fluorescent microscope, a stage with a side-view flow chamber, two 45\({}^{\circ}\) mirrors with highly reflective surface coatings (Red Optronics; Mountain View, CA, USA), a high-speed camera, and a computer (Figure 1A). There were two important modifications: (1) continuous high-intensity fluorescent light was used as the illumination source; (2) a Basler A504 kc high-speed camera was used to record the images.  
Orange tracer particles (1 \(\times\) 10\({}^{10}\) particles mL\({}^{-1}\)) with a diameter of 1 \(\upmu\)m (Invitrogen; Carlsbad, CA, USA) were diluted to 0.02% by volume with DMEM/F12 containing 1% BSA. The particle solution was then put in a 37 \({}^{\circ}\)C incubator for at least 24 h to prevent potential nonspecific aggregations. A syringe pump (Harvard Apparatus; Holliston, MA, USA) was used to drive the flow. We set two volumetric flow rates, 91.25 \(\upmu\)L min\({}^{-1}\) and 292 \(\upmu\)L min\({}^{-1}\), which equals wall shear rates of 62.5 s\({}^{-1}\) and 200 s\({}^{-1}\) respectively. The calculation followed Equation (1),  
\[\tau_{w}=\frac{6\mu Q}{WH^{2}}F \tag{1}\]  
where \(\mu\) is fluid molecular viscosity; \(Q\) is volume flow rate; \(W\) is the width of the chamber; \(H\) is the height of the chamber; and \(F\) is the correction factor for a rectangular channel with a finite aspect ratio. \(F\) is equal to 1.45 when an aspect ratio is 1.27 here [29], and the viscosity was measured to be approximately 1.5 cP at room temperature, by a RotoVisco 1 cone-plate viscometer (ThermoMC; Madison, WI, USA).  
Figure 1: Illustration of side-view micro-PIV experiment and CFD simulation. (**A**) Setup of side-view micro-PIV system (drawn by the authors); (**B**) Six relative PMN-to-TC position states (PMN: polymorphonuclear neutrophil; TC: tumor cell); (**C**) Velocity profiles around an adherent cell; (**D**) Plot of a column of velocity profile above an adherent cell calculated from micro-PIV and CFD.  
The inner microslide was coated with EI cells before being inserted into a bigger one. Cell suspension was mixed with fMLP-stimulated PMNs and Lu1205 melanoma cells at a 1:1 ratio, and soluble Fg or soluble Fn was added at the desired concentration. The mixed solution was then perfused into the side-view chamber at given experimental shear rates of 62.5 s\({}^{-1}\) and 200 s\({}^{-1}\). As described previously, six PMN-to-TC position states--(A) a single cell; (B) a TC approaches a PMN; (C) a TC collides with a PMN; (D) a TC forms a transient aggregate with a PMN; (E) a TC firmly adheres to a PMN; and (F) a TC detaches from a PMN--were chosen to be analyzed in a very low Reynolds number Stokes flow (Figure 1B). Besides bright images of adherent cells under both top view and side view, 2000 side-view PIV images were taken by a Basler A504 kc camera at a speed of 1000 fps (\(\Delta\)t is 1 ms, digital gain is 2, offset is \(\sim\)50-90, and ROI is 1280 \(\times\) 512). Images were then analyzed offline.