Magnetic separation stand

Proximal tubules were isolated as described previously^{41 (link),42 (link)} with several modifications. Briefly, mice were perfused through the thoracic aorta with 20 mL of a cold magnetic bead solution supplemented with 40 μL of Dynabeads M450 (14013, Invitrogen). Kidney cortex was collected and minced into 1-mm³ pieces, which were then digested in HBSS supplemented with 1 mg/mL collagenase I (LS004196, Worthington), 0.75 mg/mL trypsin inhibitor (T6522, Sigma), and 40 U/mL DNase I (D4513, Sigma) at 37 °C for 10 min on a rotator. This digestion step was performed twice and the suspension was passed through cell strainers from 100 to 45 μm to remove large cellular debris. Digested tissues on the cell strainer were collected with cold PBS, and washed three times in cold PBS. The filtrates were placed on a Magnetic Separation Stand (Promega) to remove glomeruli. The long segments of proximal tubules in suspension were used immediately in subsequent experiments.

20 µL of glyphosate-standard, control (diluent/zero standard solution), or beer sample solution, respectively, was pipetted into a 1.5 mL vial. In the same vial, 80 µL of assay buffer was added and the 100 µL solution was vortexed. Then, 8 µL of the diluted derivatization reagent was added and each vial vortexed immediately. Then, the derivatization reaction was allowed to proceed for 10 min. Meanwhile, the stock anti-glyphosate-IgG modified MBs suspension was mixed thoroughly. After 10 min, 200 µL of this stock MB suspension was added to each vial and briefly vortexed. After 30 min, 100 µL of glyphosate-enzyme-conjugate was added. Mixtures were vortexed and incubated for 30 min at room temperature. Each vial was then placed onto the magnetic separation stand (Promega, Milan, Italy) for 2 min to collect MB on the inner vial wall. The solution was then completely removed, and 400 µL of washing solution was added in each vial. The washing procedure was repeated twice with 200 µL and 100 µL of the washing solution, respectively, after which the vials were removed from the magnetic stand. 60 µL of the substrate solution was then added to each vial and, after 2 s of mixing, was allowed to incubate for 20 min. Finally, MBs were magnetically removed and the solution was completely transferred onto the electrochemical cell for chronoamperometric measurement.