PA spheres were generated by the methods as described33 , with some minor changes. We used the 3 µm SPG frits to extrude high stiffness PA gel into hexane at 300 RPM stirring. Beads were fixed overnight in AIBN as described33 , washed in fresh hexane, dried in air and resuspended into PBS with sonication to separate the beads. The spheres were 12–20 µm in diameter after resuspension, and were coated in a similar manner to the pads above. An aliquot of the PA beads was centrifuged in an Eppendorf tube (7000 RPM, 4 min), and resuspended in 10 mg/mL EDAC in pH 6 MES buffer and rotated for 15 min at room temperature. We centrifuged the beads to remove the EDAC as above, and resuspended them in 10 ug/mL alexafluor 488 goat anti mouse IgG (Invitrogen). After 30 min rotation, the beads were again centrifuged to remove unbound IgG, and resuspended in EDAC (10 mg/mL) with a 10−6 dilution of 0.1 µm red fluorescent microspheres (Invitrogen) in PBS. Beads were again rotated for 30 min and then pelleted, and resuspended in plain PBS.
To attach the coated beads to a PA gel pad, low concentrations of beads were suspended onto the gels within the cloning cylinders, and again EDAC was added to 10 mg/ml final concentration. The IgG fluorescence was used to find the PA beads, and then an isolated red fluorescent bead was imaged for the PSF.
To attach the coated beads to a PA gel pad, low concentrations of beads were suspended onto the gels within the cloning cylinders, and again EDAC was added to 10 mg/ml final concentration. The IgG fluorescence was used to find the PA beads, and then an isolated red fluorescent bead was imaged for the PSF.