Centrifuge 5417c

For qPCR and Sanger sequencing, the DNA template was prepared by heat lysis. To perform this, a single colony from an overnight (14 to 20 h) culture at 37 °C on nutrient agar was dissolved in 60 μl sterile deionized water and incubated at 95 °C in a heating block for 10 min. After cooling for a minimum of 10 min at 4 °C (in the fridge) and centrifugation for 10 min at 11000×g using Centrifuge 5417C (Eppendorf, Hamburg, Germany), the supernatant was stored at − 20 °C and used for further analysis.
For WGS and parts of the qPCR analysis, genomic DNA was extracted with the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, Saint Louis, USA) according to the manufacturer’s instructions.

The sedimentation speeds of AgNPs with or without Ca²⁺-mediated aggregation were compared at the same centrifugal force. AgNPs were aggregated by mixing 10 mL AgNP suspension with 10 mL Ca(NO₃)₂ solution (2 mM). After 10 min, the AgNPs/Ca(NO₃)₂ mixture was aliquoted into several centrifuge tubes (1 mL for each tube). Diluting 10 mL of the same AgNP suspension in 10 mL DI H₂O was used as control. Centrifugation was undertaken to sediment aggregates or individual AgNPs at a centrifugal force of 20,100g (Centrifuge 5417 C, Eppendorf, Engelsdorf, Germany), leaving dissolved Ag in the supernatant. To investigate the extent to which pre-aggregation can reduce centrifugation time, one tube from each treatment was taken to measure the Ag content in the supernatant (0.4–0.5 mL) at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h. The supernatants were acidified to 1 % HNO₃ (w/v) overnight (more than 12 h) at 80 °C, and further diluted to a final HNO₃ concentration of 0.2 % (w/v) for Ag concentration measurement by GFAAS.

HEK293T cells were co-transfected in a 6-well format with proviral constructs (5 µg) and an expression plasmid for the vesicular stomatitis virus glycoprotein (pHIT_VSV-G; 1 µg) (Fouchier et al., 1997 (link)) using the calcium phosphate method. Two days post-transfection, cell culture supernatants were harvested and cleared by centrifugation (1700 g, 4 min, 4°C, Centrifuge 5417C (Eppendorf, Hamburg, Germany), fixed-angle rotor F-45-30-11). For infection of primary CD4 +T cells (RNA-Seq, cytokine arrays, qRT-PCR), virus stocks were concentrated 20-fold via ultracentrifugation (96325 g, 120 min, 4°C, UC OptimaTM L-80 XP Ultracentrifuge (Beckman Coulter, Brea, CA), Swinging-Bucket Rotor SW Ti-32) and for each of the three HIV-1 strains, wild type and mutant viruses were adjusted for infectivity using a TZM-bl reporter cell assay.

HEK293T cells were co-transfected in a six-well format with proviral constructs (5 μg) and an expression plasmid for the vesicular stomatitis virus glycoprotein (pHIT_VSV-G; 1 μg) (39 (link)) using the calcium phosphate method. Two days post-transfection, cell culture supernatants were harvested and cleared by centrifugation (1700 g, 4 min, 4°C, Centrifuge 5417C (Eppendorf, Hamburg, Germany), fixed-angle rotor F-45-30-11). In some cases, virus stocks were concentrated 20-fold via ultracentrifugation (96 325 g, 120 min, 4°C, UC OptimaTM L-80 XP Ultracentrifuge (Beckman Coulter, Brea, CA, USA), Swinging-Bucket Rotor SW Ti-32) before infection of primary cells.

The remaining rotor contents were transferred with a pipette to a microcentrifuge tube containing a mixture of zirconia beads (1 mm, 167 μL or ~375 mg; 0.7 mm, 334 μL or ~1,314 mg; 500 μL total) on dry ice. The old tube was rinsed by briefly vortexing with 800 μL MeOH (80% in ddH₂O), which was added to the beads. This mixture was frozen on dry ice for up to 3 days. Contents were twice homogenized on dry ice for 180 s @1,800 rpm using a MP FastPrep 96 (MP Biomedical; USA) adapted for microcentrifuge tubes, adding dry ice each time. The homogenate was centrifuged at 14k rpm at 4°C for 5 min (18,220x g; centrifuge 5417C; Eppendorf, USA). The supernatant was transferred to a separate microcentrifuge tube and kept on dry ice while the pellets were back-extracted with 500 μL MeOH (80%), homogenized once for 180 s at 1,800 rpm, and centrifuged an additional 5 min. Supernatants from both extractions were combined, then dried to completion in a CentriVap concentrator/CentriVap cold trap −105°C system (Labconco, Kanasas City, MO, USA) for 4–6 h. Pellets for two samples were combined during resuspension in D₂O (DSS, 1/9 mM) for each condition. Two replicates from each condition were thus pooled and pipetted into 1.7 mm NMR tubes (Bruker).