Beckman sw 55 rotor

Cultured normal and fmr1 mutant lymphoblastoid cells were incubated with cycloheximide (100 μg/ml) or 30 mM EDTA at 37 °C for 30 min to arrest polyribosome migration. Cells were lysed using lysis buffer (10 mM HEPES-KOH, pH 7.5; 150 mM KCl; 10 mM MgCl₂; 1 mM DTT; 100 μg/mL cycloheximide; and 1% Triton X-100, supplemented with proteinase and RNase inhibitors). Cytoplasmic extracts were loaded on a 15–60% (wt/vol) sucrose gradient and centrifuged at 45,000 rpm in a Beckman SW-55 rotor (Beckman Coulter, CA, USA) for 60 min at 4 °C to separate them into 10 fractions. Each fraction was isolated and analyzed by RT-qPCR [33 (link)], with an equal amount of C. elegans RNA used as an external control.

All the viruses were mass produced in EPC cells and supernatants of the infected cells were clarified by low speed centrifugation (4000 rpm 15 min). Supernatants (35 mL) were first concentrated by ultracentrifugation in a SW28 Beckman rotor (Beckman Coulter, Villepinte, France) at 25,000 rpm for 90 min at 4 °C, resuspended in cell culture medium without fetal serum and then loaded on a 25% sucrose cushion in TEN buffer 1× (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8). After ultracentrifugation at 36,000 rpm in a SW41 Beckman rotor (Beckman Coulter, Villepinte, France) for 4 h at 4 °C, viral pellets were resuspended in 100 μL of TEN, loaded onto a 15%–45% discontinuous sucrose gradient, and ultracentrifuged overnight at 25,000 rpm in a Beckman SW55 rotor (Beckman Coulter) at 4 °C. Unique bands of purified viral particles were collected, diluted in TEN and pelleted by ultracentrifugation in a SW41 Beckman rotor at 25,000 rpm for 90 min at 4 °C. Final pellets were resuspended in TEN (60 μL) and stored at −80 °C until further use.