This protocol observes and analyzes cell rolling trajectories on asymmetric receptor patterns to facilitate engineering of receptor pattern substrates for label-free cell separation and analysis begin by micro contact printing of PEG molecules on a gold substrate to create alternating patterns of self-assembled monolayers, followed by backfilling the substrate with P select and receptors. Next, incorporate the substrate with the flow chamber. Flow the HL 60 cells into the chamber.
Record an image sequence of the rolling interaction of HL 60 cells on P selectin patterns and analyze the images converted from the video. The trajectories of rolling cell edge tracking length rolling velocities on plane, region, and edge can be obtained using a MATLAB program. This method can provide insight into how HO 60 cells move on the as symmetric receptor patterns in share flow.
This can be applied for quantifying other cells surface interactions, direct analysis of the cells by observing their trajectories on receptor patterns. And for design of micro footing devices for cell separation. Micro contact printing is used to create alternating patterns of self-assembled monolayers of peg molecules on the gold substrate.
First ink the PDMS stamp with five millimolar PEG solution in ethanol. Dry the surface for 20 minutes. Gently put the stamp on the gold surface for 40 seconds and make sure there is a good contact between the gold surface and the stamp.
No excess pressure is required. Rinse the surface with ethanol and dry under a stream of nitrogen. Now using a perfusion chamber, incubate the substrates with 15 micrograms per milliliter P selectin solution for three hours at room temperature to pattern the remaining areas with P selectin, rinse the chamber, then backfill the surfaces with one milligram per milliliter, BSA for one hour to block nonspecific interactions.
Rinse the surface with DPBS buffer before performing the cell rolling experiment. Assemble the flow chamber and then flow a suspension of 10 to the fifth cells per milliliter. HL 60 cells over the pattern, surfaces in a rectangular flow chamber with inclination angles of the receptor pattern of 10 degrees.
At room temperature of 24.5 degrees Celsius. Adjust the syringe pump to generate a flow rate of 75 microliters per minute with corresponding laminar shear stress of 0.5 dines per centimeter squared. Now with an inverted microscope and mounted camera record HL 60 rolling.
Interactions with adhesive p, select and substrates. Using a four x objective at a rate of one frame per second for durations of 300 seconds. Perform three independent experiments.
Present the data as mean and standard deviation of the average values obtained from each experiment. Microscope images converted from the video of HL 60.Rolling. Interactions with adhesive p, select and substrates demonstrate bright and dark regions corresponding to p selectin receptor and PEG regions respectively.
Tracks are delineated using a customized MATLAB program here. The edge inclination angle was 10 degrees and the shear stress was 0.5 dines per centimeter squared. Further analyses form on the distribution of edge tracking length, the average value of on edge tracking length, as well as VE and VP, the rolling velocities on the edge and inside of the bands.
After watching this video, you should have a good understanding of how to examine the cell trajectories on the asymmetric receptor patterns.
