β casein

Protoplasts were loaded in a custom-built chamber, which was passivated with 0.2 mg/ml β-casein (Millipore-Sigma, Saint-Louis) for 30 min and pre-equilibrated with EMM5S supplemented with 0.8 M D-sorbitol. A glass micropipette (#1B100-4; World Precision Instruments, Sarasota) was forged to a diameter smaller than the average protoplast radius (~2.5 µm) and was connected to a water reservoir of adjustable height to apply a defined aspiration pressure. Before and after each experiment, the height of the water reservoir was adjusted to set the aspiration pressure to 0. Cells were imaged with a bright-field IX-71 inverted microscope (Olympus, Tokyo, Japan) equipped with a 60x/1.4NA objective, and images were recorded every second. Aspiration pressure was gradually increased every 30 s and the membrane tension $\sigma$ was calculated as $\sigma =∆P.R_p/\left[2\right(1-R_p/R_c\left)\right]$ , where $R_p$ and $R_c$ are, respectively, the micropipette and the cell radius, $∆P$ is the aspiration pressure for which the length of the tongue of the protoplast in the micropipette is equal to $R_p$ (Evans and Yeung, 1989 (link)). To limit the effects of the adaptation of cells’ membrane tension, all measurements were performed within the first 5 min after the hypotonic shock, which greatly limited the throughput of our assay (one measurement per sample), compared to the measurements at steady state (around six measurements per sample).

A sample chamber consists of a flow cell sandwiched between two microscope coverslips coated with a thin layer of nitrocellulose by spin coating^{21 (link)}. Prior to chamber assembly, coated coverslips were rinsed and stored in a clean plastic container for 48 h or more before being assembled into a microfluidic sample chamber using inert silicone high vacuum grease (Dow Corning 1597418). In contrast to previous work, nitrocellulose-coated coverslips were used for both the top and bottom surfaces of the sample chamber instead of only the bottom surface.
Tethers for MT experiments were formed in a sample chamber^{21 (link),22 (link)}. First, a nitrocellulose coated sample chamber was incubated with 10 ng/µL anti-digoxygenin (Vector Labs MB-7000) in phosphate buffered saline for 30 min. Next, 1 µm magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher 65601) coated with biotin and digoxigenin labeled DNA were bound to the surface to serve as fiducial markers. Then, 5 mg/ml β-casein (MilliporeSigma C6905) was incubated in the chamber for at least 1 h to passivate the surfaces. The DNA substrate of interest was then incubated at concentrations ranging from 1.5 pM to 10 pM for 15 min, followed by magnetic beads at a concentration of 20 µg/ml for 15 min. The buffer in the sample chamber was then exchanged for the topo reaction buffer.

Powdered CdCl₂, Cr₂O₃, PbCl₂, and β-casein were acquired from Merck. The dairy matrix (whole milk) in pasteurized form was obtained from a local brand (Santa Clara^® owned by the Coca-Cola^® group) and stored at 4 °C for preservation.