Jla 10.500 rotor

Vero cells were grown to 80% confluency in ten 175 cm² flasks and infected with RVFV virus at a multiplicity of infection (MOI) equal to 0.1. At 72 hours post-infection (p.i.), supernatants were harvested and cellular debris removed by centrifugation in a JLA 10.500 rotor (Beckman Coulter, Fullerton, CA) (4°C, 10 minutes, at 2600×g). Virus was precipitated from the clarified supernatant by adding polyethylene glycol (PEG) to a final concentration of 5% and stirring the solution overnight at 4°C. The precipitated virus was pelleted by centrifugation in the JLA 10.500 rotor (4°C, 30 minutes, at 2600×g), and the supernatants were decanted and viral pellets resuspended in a minimal (∼1 ml) volume of sterile PBS. Viral suspensions were applied to sterile 10–60% sucrose gradients and ultra-centrifuged for two hours at 32,000 rpm (126,000×g) in a SW-41 swinging bucket rotor (Beckman Coulter, Fullerton, CA). A distinct band corresponding to RVFV was removed and re-centrifuged overnight at 22,000 rpm in a JA 20 rotor (Beckman Coulter, Fullerton, CA) to pellet the virus. The supernatants were discarded and the RVFV was resuspended in a minimal volume of PBS, separated into aliquots and frozen at −80°C until analysis.

Cell cultures in late exponential growth were collected by centrifugation at 5000 rpm for 15 min at 4°C in a JLA-8.1000 rotor (Beckman Coulter, High Wycombe, UK). Cell pellets were resuspended in one-quarter culture volume of wash buffer (10 mM NaPO₄ pH 7.5 and 0.5 mM EDTA) and then washed three times by centrifugation at 5000 rpm for 15 min at 4°C in a JLA-10.500 rotor (Beckman Coulter) and resuspension in the same volume of wash buffer. After the final centrifugation, the cell pellet was weighed and resuspended in wash buffer, using a ratio of 0.3 mL wash buffer per gram of cell pellet. The cell suspension was then quick-frozen by drop-wise addition into liquid nitrogen and stored at −80°C until further use.
Cryogrinding was performed using an RM100 electric mortar grinder with a zirconium oxide mortar and pestle (Retsch, Hope Valley, UK). The mortar and pestle were pre-cooled by filling with liquid nitrogen for 10 min before grinding. Frozen cells were then added into the pre-cooled grinder and ground for 40 min, with regular generous addition of liquid nitrogen to maintain the temperature and prevent cell clumping during the grinding process. Cryogrindate cell powder was recovered and stored at −80°C until further use.

Lignin composition, as well as monomer linkages, was determined by 2D HSQC NMR analysis [32 (link)]. Milled samples (100 mg) were dissolved in 3.6 mL of DMSO and 1.8 mL of N-methylimidazole, and acetylated by addition of acetic acid for 4 h at room temperature. Samples were then precipitated in water, and centrifuged at 18,600g for 10 min (Beckman JLA-10.500 rotor). The pellets were washed twice and centrifuged again as described above. Acetylated samples (80 mg) were dissolved in 0.6 mL CDCl₃ in a 5-mm NMR tube. NMR spectra were acquired and treated as described previously [33 (link)].