Isolation of Glycosomes from Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles of trypanosomatids in which the glycolytic and some other metabolic pathways are compartmentalized. We describe here two methods for the purification of glycosomes from Trypanosoma cruzi for preparative purposes, differential and isopycnic centrifugation. These are two techniques that allow the separation of different cellular compartments based on their different physicochemical characteristics. The first type of centrifugation is a rapid method that does not require large inputs and allows for fractions enriched in specific cell compartments to be obtained. The second type of centrifugation is a more elaborate method, but enables highly purified cellular compartments to be isolated. The success in obtaining these purified, intact organelles critically depends on using an appropriate method for controlled rupture of the cells.

Effect of rotor type on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

Separation of differentially isotope-labeled bacterial RNA by isopycnic density gradient centrifugation is a critical step in RNA-based stable isotope probing analyses, which help to link the structure and function of complex microbial communities. Using isotope-labeled Escherichia coli RNA, we showed that an 8 mL near-vertical rotor performed better than a 2 mL fixed-angle rotor, thereby corroborating current recommendations. Neither increased concentrations of formamide nor urea in the medium improved the separation results using the fixed-angle rotor.

Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs.

Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic "Lab-on-a-Disc" cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are 'low-pass', i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both 'hydrostatically' and 'hydrodynamically' triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, 'dual siphon' configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.

Purification of peroxisomes using a density barrier in a swinging-bucket rotor.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a preformed isosmotic continuous gradient. Because of the large difference in density between peroxisomes and the next densest organelle (mitochondria), a density barrier is effective. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll, there is no contamination from endoplasmic reticulum.

Purification of viruses by centrifugation in gradients of inert substances.

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in "self formed" caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation.

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid method of isolation and fractionation of proteoglycans using a vertical tube rotor for isopycnic centrifugation.

A comparison was made between a vertical tube rotor and a fixed angle rotor for isopycnic centrifugation of proteoglycans. In the vertical tube rotor, isopycnic gradient was achieved much faster than in the conventional fixed angle rotor. The use of a vertical tube rotor for isopycnic centrifugation shortens the time considerably for the isolation of proteoglycans from various tissues.

The applicability of freeze-thaw Percoll gradients to whole-cell isopycnic fractionations: preliminary results.

The freeze-thaw method of gradient generation, using Percoll as the density medium, is shown to be adaptable for the isopycnic sedimentation of live whole cells. Methods of controlling gradient profiles are examined with preliminary evidence showing that three types of cells brought to isopycnic equilibrium in these gradients and recovered from them retain satisfactory levels of several vital functions.