g-Max System Complements Beckman Coulter Ultracentrifuge Rotors

The ultracentrifuge rotors from Beckman Coulter can be enhanced by adding the g-Max system. The capabilities this adds include flexibility of volumes, high efficiency and heat-sealed technology. This article explains the advantages of the g-Max system and also describes a technique to significantly reduce lab time using the system.

Flexibility of Volumes

Owing to an innovative approach to tube support in the rotor cavities, the g-Max system allows for the running of smaller volumes in fixed angle, vertical and swinging bucket rotors, providing increased efficiency, with no decrease in  centrifugal acceleration. Floating spacers and Quick-Seal bell-top polypropylene tubes patented by Beckman Coulter are used in this novel system.  

In contrast to traditional sleeve-type adapters, the g-Max spacers "float" on top of the tube, thereby ensuring the sample is kept at the maximum radius of the tube cavity. This allows smaller volumes to be run in the rotors, with higher efficiency.

Maximized Efficiency

Separations take less time for pelleting applications. g-Max technology enables considerable time-saving and higher efficiency by minimizing the path length a particle needs to travel prior to pelleting at the bottom of the tube.

Heat-Sealed Technology

Quick-Seal tubes, available in many sizes and 3 different designs (bell-top, dome-top, and conical bell-top), have been developed for use in all swinging bucket, near-vertical tube, vertical tube, and most fixed-angle rotors. For runs where a potential contamination risk exists due to biohazards, Quick-Seal tubes are particularly useful. The need for caps is eliminated because the tubes are heat-sealed to provide a highly reliable seal, meaning scientists are kept safe and the environment is kept clean.

Fixed-Angle Rotors

How g-Max technology compares to both a traditional adapter and a full-size tube is shown in Figure 1. In comparison with full-size tubes, the path length of the samples in the g-Max System in fixed angle rotors is shorter, while maximum radius stays the same. Pelleting time is reduced and maximum g-forces are retained. By contrast, in the conventional sleeve adapter, the tube is moved in from the maximum radius, leading to a reduction in g-force and no significant time saving.

Figure 1. The g-Max System in fixed-angle rotors provides significant advantages over both full-size tubes and conventional adapters. Image credit: Beckman Coulter

Life Sciences

The k-factor is a good measure of tube or rotor efficiency. The k-factor is a guide to the time (t), in hours, needed to pellet a particle of a known sedimentation coefficient (s), in Svedberg units, where t = k/s.

The k-factor is directly proportional to run time and as the k-factor is lowered, the rotor efficiency increases.

For the  Type 70.1 Ti and Type 90 Ti rotors, Table 1 demonstrates an example of the higher performance offered by the g-Max system, when compared with a full size tube or a traditional adapter. Comparison of the k-factors shows that the use of  g-Max technology would mean pelleting operations take between one third and one fourth of the time taken when the sleeve-type competitor is used.   Moreover, due to stability issues, many sleeve type traditional adapters cannot be used to maximum rotor speed. In addition, the g-Max System offers savings on run times when compared with full-size tubes.

Vertical Tube Rotors

Since vertical tube rotors enable short run times, they are frequently used for gradient separations. Two or more tube sizes can be run by all Beckman Coulter vertical tube rotors by incorporating the g-Max system. Plasmid DNA preparations are one type of separation that is commonly performed in these rotors. Although the g-Max system will not impact on run times due to there being no change in path length, the technology allows for volume flexibility in vertical rotors, which can help save critical samples or reduce a large dilution effect  (Figure 2).

Table 1. Performance Comparisons by Rotor Type.

Figure 2. Vertical tube rotors are also compatible with g-Max tubes, providing flexibility of volumes to your experimental design. Image credit: Beckman Coulter

Swinging Bucket Rotors

A key benefit of the g-Max system is secondary containment, particularly in swinging bucket rotors. The quick-seal tubes are especially useful for biohazardous radioactive applications, owing to the fact that they have a highly reliable seal instead of cap.  In cases where large interband volumes are not critical, using short path lengths, as allowed by the g-Max floating spacers, allows for some of the best time savings for separations. As shown in Figure 3, the small path length tubes offer shorter run times, volume flexibility and reduced interband volumes.

Figure 3. Swinging bucket rotors offer the most significant advantages with the g-Max System. Image credit: Beckman Coulter

Benefits Overview of Rotor Type

The benefits of the  g-Max System compared with common tubes and sleeve adapters applies to all rotor types, although the greatest advantages are observed in the swinging and fixed-angle bucket rotors. These benefits are listed in Table 2 for the three main rotor types.

Table 2. Benefits of g-Max System by Rotor Type

Example of Time Savings: Exosome Purification

Rapid expansion of research into exosomes has led to a  large increase recently in the number of publications on the subject. These small vesicles, which are  between 30 and 100nm in size, have been shown to be released by many cell types, exhibiting a range of functions and often playing a role in cancer.

To further advance this exciting field, an enhanced and more efficient protocol for isolating exosomes is vital. A technique is presented here for obtaining a high-purity, high yield collection of exosome vesicles using a method that employs g-Max technology.

In order to remove dead cells and debris, the pelleting and centrifuging of cultured cells is usually performed in two to three steps, at a low speed. Then, exosomes and contaminating proteins are pelleted over an extended, 90-minute period using an ultracentrifuge at speeds of 100,000 x g.

Next, the pellet is resuspended in buffer (normally PBS) and layered on top of a density gradient and spun for 18h. Fractions are collected, each pelleted separately by 2 further spins at 100,000 x g for 1 h and then characterized by size particle analyzers, electron microscopy, or Western blots so that size and purity can be determined. This is a time-consuming and tedious technique, although it does generate  high purity exosomes of proper size and shape.

Figure 4 shows how path length can be shortened while high g-forces are retained, using advanced g-Max technology. This method provides significant savings on lab time.

Figure. 4. Comparative protocols showing critical time savings using g-Max technology. Over 1 hour of time was saved in the centrifuge using the modified protocol. Image credit: Beckman Coulter

Revised Method

In this  revised protocol, the Vi-CELL from Beckman Coulter was used to determine the viability of cells cultured from frozen stock.  Using the Coulter Principle, the Vi-CELL offers precise count,  sizing,  and viability of cells in culture.

After acquiring highly viable cells, 50mL conical tubes were filled with 25mL of cell culture samples (6 x 10₅ cells/mL), and placed in a SX4750A rotor equipped with 50mL conical adapters in the Allegra X-15R tabletop centrifuge. The tubes were spun at 750 x g for 10mins. The supernatant was then recovered and spun for 20mins at 2,000 x g. Using the Optima XPN ultracentrifuge  with a SW 32 Ti rotor, the supernatant was spun again for 20mins at 10,000 x g using Quick-Seal 15 mL tubes, to eliminate cell debris. The supernatant was recovered again, filtered through a 0.22µm membrane and spun for 40 mins at 100,000 x g in15mL Quick-Seal tubes with g-Max adapters. The supernatant was then aspirated and the pellet was recovered by resuspension in 1x PBS.

The Biomek 4000 Laboratory Automation Workstation from Beckman Coulter provides a consistent, fast and reproducible technique for layering a density gradient with the density and volume shown in Figure 5. The resuspended exosomes (1mL) were then layered on top of the density gradient and spun for 18h at 100,000 x g, at 4°C.

Figure 5. Beckman Coulter's Biomek 4000 Workstation (a) was used to gently layer four increasingly dense Opti-Prep solutions (b). Food coloring dye was used to show the layering (c) in a centrifuge tube using this gentle and reproducible technique. Image credit: Beckman Coulter

For a total volume of 1.5 mL per combined fraction, 500µl fractions were obtained from the top and combined with every third fraction. Using Quick-Seal 1.5mL tubes with g-Max adapters, the samples were spun at 4°Cin the Optima MAX-XP ultracentrifuge for 45mins at 100,000 x g. To remove any excess Opti-Prep, the pellet was resuspended in PBS and spun in the Optima MAX-XP ultracentrifuge for a further 45mins at 100,000 x g using the same 1.5mL Quick-Seal tubes with g-Max adapters. Before conducting purity analysis, the final pellet was again suspended in 100µl PBS and frozen at -20°C.

Figure 6 shows how the DelsaMax System was used to characterize particle size between the two methods. For the general, published protocols for separation as well as the revised g-Max procedure, all eight 1.5mL fractions were analyzed using DLS with rigid parameters. As shown by peaks that were less than 10nm in diameter (data not shown), early fractions 1 to 12 had high protein contamination. The later fractions 16 to 24 also had high protein contamination as well as containing particles that were more than 250nm in diameter, indicating contamination with cell debris or other large molecules  (data not shown).

Figure. 6. DelsaMax characterization of resuspended pellets, following density gradient separation, where the particle size using g-Max technology (red) (n=9) is compared to the published protocol (black) (n=3). Both protocols generate particles of size less than 20 nm suggesting some protein contamination. However, particles between 30 nm and 100 nm represent the appropriate size of exosomes. Image credit: Beckman Coulter

Conclusions

For both the protocols, fractions 13 to 15 contained particles of size 30 to 100nm diameter, indicating exosomes. This shows that the exosomes were properly isolated from contaminants by the density gradient and that the protocols are similarly efficient at isolating exosomes. This data supports our technique and the use of g-Max technology to produce accurate and reproducible results, while saving on critical time.

References

1. U.S. Patent Nos. 4,301,963; 4,304,356; 4,290,550; British Patent No. 2,021,982; Canadian Patent No. 1,132,509; Japanese Patent No. 1,469,153; Italian Patent No. 1,121,772.
2. Keller S, Sanderson MP, Stoeck A, Altevogt P. Exosomes: from biogenesis and secretion to biological function. Immunol Lett. 107(2); 102–8: (2006). doi:10.1016/j.imlet.2006.09.005
3. Duijvesz D et al. Exosomes as biomarker treasure chests for prostate cancer. European urology. 59.5; 823–831: (2011).
4. Iero M, Valenti R, Huber V, Filipazzi P, Parmiani G, Fais S, et al. Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ. 15(1); 80–8: (2007). doi:10.1038/sj.cdd.4402237
5. Yang C, Robbins PD. The roles of tumor-derived exosomes in cancer pathogenesis. Clin Dev Immunol. 2011; 1–11: (2011). doi:10.1155/2011/842849
6. Azorsa DO. The genomic and proteomic content of cancer cell-derived exosomes. Front Oncol. 2; 38: (2012). doi:10.3389/fonc.2012.00038/abstract
7. Simpson RJ, Mathivanan S. Extracellular microvesicles: the need for internationally recognised nomenclature and stringent purification criteria. J Proteomics Bioinform. 5: (2012).
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9. Tauro BJ et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 56.2; 293–304: (2012).

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