Polysome profiling was modified from (Masek et al., 2011 ). C. albicans cultures were grown to OD600 = 0.6, treated with 1 mg ml−1 cycloheximide, and incubated at 4°C for 5 min. Fifty milliliters of culture were collected for each sample. Extraction buffer was supplemented with 1 mg ml−1 cycloheximide. 7–47% sucrose gradients were prepared by stepwise freezing of 2.4 ml each of 7%, 17%, 27%, 37% and 47% sucrose in Beckman Coulter polyallomer centrifuge tubes at −80°C, then thawing at 4°C overnight. For EDTA control samples, each gradient layer contained 20 mM EDTA. Lysate samples were applied to gradients and centrifuged at 35,000 r.p.m. at 4°C for 2.5 h in a Beckman SW41 rotor. Gradients were fractionated, absorbance was read at 254 nm, and polysome profile was created using the ISCO Gradient Fractionator (Teledyne). Fractions were collected using the Foxy Jr Fraction Collector. Fractions of 500 μl each were added to 800 μl 8 M guanidine HCl and 700 μl 100% EtOH, and placed at −20°C overnight for RNA precipitation. Pellets were washed with 70% EtOH and resuspended in 30 μl RNase‐free water for subsequent use in RT‐qPCR.
Polyallomer centrifuge tube
Manufactured by Beckman Coulter
Sourced in United States
Polyallomer centrifuge tubes are laboratory consumables designed for use in centrifugation processes. They are made of a durable polyallomer material and are available in various sizes and shapes to accommodate different sample volumes and centrifugation needs.
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Lab products found in correlation
Isco gradient fractionator, by Teledyne (2 mentions)
Sw41 rotor, by Beckman Coulter (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Sucrose, by Beckman Coulter (1 mentions)
Sucrose, by Merck Group (1 mentions)
Optima l 90k, by Beckman Coulter (1 mentions)
Ultra clear centrifuge tube, by Beckman Coulter (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
Sw40ti, by Beckman Coulter (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Airfuge ultracentrifuge, by Beckman Coulter (1 mentions)
Anti rabbit igg conjugated with peroxidase, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Optima xe 90, by Beckman Coulter (1 mentions)
Airfuge centrifuge, by Beckman Coulter (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Linear polyacrylamide, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
26 protocols using polyallomer centrifuge tube
1
Polysome Profiling of Candida albicans
2
Lentivirus Vector Production Protocol
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LV vectors were produced as described previously.12 (link) Briefly, 1.8 × 107 HEK293T cells were plated per 15 cm sterile culture dish and transfected with the following components: 40 μg of the relevant transfer plasmid, 20 μg of pMDLg.RRE, 10 μg of pRSV-Rev, and 10 μg of pMDG.2 (all plasmids produced by PlasmidFactory). Additionally, 10 μg of pCMV-Tat (kindly provided by Professor Axel Schambach from Hannover Medical School40 (link)) was supplemented for enhanced vector titers. The plasmid mixtures were added to 5 mL Opti-MEM and filtered through 220 nm sterile filter units. Filtered DNA was combined with 5 mL Opti-MEM (Life Tech/GE) containing 2 μM polyethylenamine (PEI, Sigma). The resulting 10 mL mixture was incubated at room temperature for 10 min before addition to HEK293T cells. After 4 h, the transfection mixture was replaced with fresh culture medium. Virus supernatant was collected at 48 h and 72 h post-transfection. After each harvest, the collected medium was filtered through a cellulose acetate membrane (0.45 mm pore). LV harvests were combined before concentration by ultracentrifugation. Briefly, viruses were placed in polyallomer centrifuge tubes (Beckman Coulter) and centrifuged for 2 h at 90,000 × g at 4°C in a Sorvall Discovery 90SE Centrifuge. Following centrifugation, the supernatant was removed and pellet recovered in 200 μL Opti-MEM.
3
Sucrose Density Gradient Formation
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Density gradients were prepared according to an established procedure67 (link). Gradients were formed by dissolving 10% and 40% sucrose (Sigma-Aldrich), in 25 mM HEPES pH 7.5, 75 mM KCl, 75 mM NaCl. Gradients were set up in polyallomer centrifuge tubes (Beckmann) by filling them to half height with 40% sucrose and topping them up with an equal amount of 10% sucrose. Gradients were formed by tilting the tubes horizontally for 3 h at room temperature and then tilting them back to vertical position. Tubes were stored overnight at 4 °C and samples were loaded as described for each experiment.
4
Ultracentrifugation for Extracellular Vesicle Isolation
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For each cargo loading condition 45 ml of cell conditioned media was processed with an ultracentrifugation protocol adapted from (Osteikoetxea et al., 2015 (link)). Cell viability at time of EV isolation was 95.4%±2.1% (mean ± SD). Cell‐conditioned media was first centrifuged at 300 × g for 10 min to pellet cells and then at 2500 × g for 15 min to pellet dead cells and larger EVs. Remaining supernatant was transferred to 38 ml polyallomer centrifuge tubes (Beckman Coulter) and centrifuged in an Optima L‐90K ultracentrifuge (Beckman Coulter) using a SW32 Ti rotor (k‐Factor 204), at 16,000 × g for 30 min to deplete for remaining large and intermediate size EVs. Then a 100,000 × g spin for 70 min was used to pellet EVs for this study. The supernatant was discarded and pellets were collected in 1.5 ml PBS and re‐pelleted at 100,000 × g using a SW32 Ti rotor. The resulting pellet was re‐suspended in a final volume of 200 μl of PBS followed by aliquoting for downstream experiments and storage at ‐80°C until use. For control experiments with additional purification by size exclusion chromatography (SEC), 150μl of the final EV isolates were applied to qEVsingle / 70 nm columns (IZON Cat# SP2) and manufacturer's instructions were followed to separate an initial 1 ml void fraction, and subsequently collect 10 consecutive 0.5 ml fractions.
5
Coverslip Centrifugation Experiments
6
Isolation of Extracellular Vesicles from MSC
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MSC of three donors were cultured in three T175 cm2 flasks until 90% confluency with or without IFNγ. They were then washed with PBS and cultured for 24 h in 15 mL serum-free medium (MEM-α). Conditioned medium (45 mL) was then collected and floating cells and cellular debris removed by centrifugation at 300× g for 30 min. Subsequently, the supernatant was ultracentrifuged in polyallomer centrifuge tubes (Beckman Coulter) at 100,000× g for 2 h using a Beckman Coulter ultracentrifuge (Beckman Coulter Optima L-90K ultracentrifuge; Beckman Coulter, Fullerton, CA, USA) with a swing angle rotor type SW40Ti, similar as described before [35 (link)]. EV were collected in 200 μL of filtered PBS and stored at −80 °C until use.
7
Buoyant Density of zot-encoding Elements
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The buoyant density of the zot-encoding elements (VAIϕ) was examined by cesium chloride (CsCl) gradients of mitomycin C treated cultures. V. anguillarum strains PF4 and T265 were cultured in MB overnight. Then, the cultures were diluted 1:3 in MB +/− mitomycin C (final concentration: 50 ng mL−1: Sigma-Aldrich, St. Louis, MO, USA) and incubated under standard conditions for 24 h. Supernatants were 0.2 µm-filtered and checked for sterility by plating. The supernatants of each strain were concentrated by ultracentrifugation in Polyallomer centrifuge tubes (Beckman Coulter, Brea, CA, USA) in a Beckman Optima™ LE-BOK Ultracentrifuge using the SW 55 TI rotor at 40,000 rpm for 1 h at 4 °C. Then, the upper 80% of the supernatant was discarded, the tubes were refilled with supernatant and centrifuged as described above. This step was repeated until a total of 102 mL supernatant was concentrated to 6 mL. Then, 1 mL of the supernatant concentrates was loaded onto gradients containing fractions of 1.2–1.6 g mL−1 CsCl and centrifuged at 50,000 rpm for 20 h at 4 °C. Finally, fractions were collected in 0.4 mL steps using a 27 G needle (BRAUN, Melsungen, Germany) and the densities were determined geometrically. Each fraction was then diluted tenfold and 1 µL was used as a template in a PCR reaction targeting zot (Table S1 ).
8
Sucrose Gradient Fractionation of Proteoliposomes
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50 μl of proteoliposomes in 40% (w/v) sucrose were put into the bottom of polyallomer centrifuge tubes (Beckman Coulter) and sequentially overlaid with 100 μl of 20% sucrose solution and 50 μl of 5% sucrose solution, forming a discontinuous gradient from bottom to top. The samples were centrifuged at 100,000 g for 60 min at 4°C in an Airfuge Ultracentrifuge (Beckman Coulter) using the A-100/18 rotor. Fractions of 40 μl each were collected from top to bottom and protein was detected by western blotting.
Samples were run in 10% or 15% polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with anti-KtrB polyclonal antibody (overnight incubation at 4°C) or anti-KtrA polyclonal antibody (2 h at room temperature), respectively. Detection was done by incubation with anti-rabbit IgG conjugated with peroxidase (Sigma) for 30 min at room temperature and using Amersham ECL Prime western blotting detection reagents.
Samples were run in 10% or 15% polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with anti-KtrB polyclonal antibody (overnight incubation at 4°C) or anti-KtrA polyclonal antibody (2 h at room temperature), respectively. Detection was done by incubation with anti-rabbit IgG conjugated with peroxidase (Sigma) for 30 min at room temperature and using Amersham ECL Prime western blotting detection reagents.
9
Extracellular Vesicle Isolation Protocol
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Three hundred mL of cell cultured conditioned media was harvested from LNCaP, PC-3, and PNT2 cells at 80% confluence, and centrifuged at 1,000 x g at 4 °C for 10 min to remove remaining cells and cellular debris. The remaining supernatant was centrifuged at 2,500 x g for 25 min at 4 °C to pellet larger vesicles such apoptotic bodies. The supernatant was transferred to new tubes and centrifuged at 20,000 x gavg for 25 min at 4 °C to pellet the MV-enriched fractions. The final supernatant was ultracentrifuged at 110,000 x gavg, for 2 h using an Optima XE 90 ultracentrifuge, 45 Ti rotor and polyallomer centrifuge tubes (Beckman Coulter) k-factor 191.3, to pellet the EXO-enriched fractions. All vesicle pellets were resuspended in PBS or lysis buffer depending on the down-stream analysis and stored at -80 °C.
10
Polysome Profiling of C. albicans
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