Sav is produced in high concentrations, which allows one to set up two catalysis experiments (200-μl final volume) from a 6-ml cell culture (corresponds to ∼0.25 ml of cell lysate).
The root slices were placed in tissue culture cluster, and 1 mL of cell suspension was added carefully over the root slice.
For each experiment, 15 mL of cell suspension was prepared at the desired concentration of biomass.
Cells from 1 mL of cell culture were collected by centrifugation (13,000×g, 4°C, 10 min).
After 12 days, 10 mL of cell culture were concentrated by centrifugation at 10,000 rpm for 10 min.
After separation, approximately 0.1 mL of cell walls were added to sterile Petri dishes and covered with modified MRS agar.
After transfection, 10 mL of Cell CountingKit-8 solution (#C0038, Beyotime, Jiangsu, China) was added to the appropriate test wells and incubated for 1 h.
Saos-2 cells were then seeded onto each surface at a concentration of 3 × 104 cells mL−1 (1 mL of cell suspension per well).
Thereafter, 1 mL of cell suspension was centrifuged (13,000 rpm, 5 min) in these reaction vessels and again dried overnight at 60 °C.
After approximately 22 h, 1.5 mL of cell culture were harvested from each flask and aliquots from each culture were taken and used for preparation of EM grids.
Then, 1 ml of cell lysis solution (Beyotime, Shanghai, CHA) was added to the mortars, and the samples were further ground on ice for another 5 min.
