For adhesion on coverslips, 50 µl of 0.1–10 mg/mL Poly-L-lysine Hydrobromide (>70k MW, MP Biomedicals, or 70k-150k MW, Sigma-Aldrich) is mixed with 7.5% sodium bicarbonate to achieve a pH 9, and then is placed on coverslip within a chamber, and sandwiched with another coverslip which was treated with Rain-X to spread the PLL across the entire surface for 15 min. The coverslip is then rinsed with water, and then with O-buffer.
For high adhesion on polyacrylamide gels, the gels are polymerized onto cleaned coverslip surfaces. The coverslips are treated with glutaraldehyde and then aminopropyl silane which will react with the acrylamide. Different concentrations of bis/acrylamide are mixed with 0.1 mg/mL ammonium persulfate to yield a gel with variable Elastic Modulus (ν = 0.5). Within the gel, 40 nm far red (647 nm) beads are embedded prior to polymerization. 12 µl of gel solution is then added to the coverslip and covered with a Rain-X-treated coverslip to make it hydrophilic. After the gels are polymerized, they are reacted with by the Sulfo-Sanpah protocol as previously published25 (link). The surface of the reactive gels is then coated with Poly-L-lysine Hydrobromide of »70k MW (MP Biomedicals) which has been re-suspended in MilliQ water, and the pH set to 9. The reaction proceeds for 1 hour in the dark, and the coverslips are then rinsed in 1X PBS.
For high adhesion on polyacrylamide gels, the gels are polymerized onto cleaned coverslip surfaces. The coverslips are treated with glutaraldehyde and then aminopropyl silane which will react with the acrylamide. Different concentrations of bis/acrylamide are mixed with 0.1 mg/mL ammonium persulfate to yield a gel with variable Elastic Modulus (ν = 0.5). Within the gel, 40 nm far red (647 nm) beads are embedded prior to polymerization. 12 µl of gel solution is then added to the coverslip and covered with a Rain-X-treated coverslip to make it hydrophilic. After the gels are polymerized, they are reacted with by the Sulfo-Sanpah protocol as previously published25 (link). The surface of the reactive gels is then coated with Poly-L-lysine Hydrobromide of »70k MW (MP Biomedicals) which has been re-suspended in MilliQ water, and the pH set to 9. The reaction proceeds for 1 hour in the dark, and the coverslips are then rinsed in 1X PBS.