Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes
The suitability of Iodixanol {5,5′-[(2-hydroxy-1, 3-propanediyl)-bis(acetylamino)] bis-[N,N’-bis(2, 3-dihydroxypreopyl-2,4,6-triiodo-1,3-benzenecarboxamide)]}, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) Iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient. Peroxisomes banded around 30% (w/v) Iodixanol (d approximately 1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of Iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.
Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood
Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells.
To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties.
We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.
Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes
The suitability of Iodixanol {5,5′-[(2-hydroxy-1, 3-propanediyl)-bis(acetylamino)] bis-[N,N’-bis(2, 3-dihydroxypreopyl-2,4,6-triiodo-1,3-benzenecarboxamide)]}, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) Iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient.
Peroxisomes banded around 30% (w/v) Iodixanol (d approximately 1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of Iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.
A new, fast and convenient method for layering blood or bone marrow over a density gradient medium
A density gradient dispenser has been developed, providing a fast, easy and convenient method for layering peripheral blood or bone marrow over a density gradient solution for the isolation of lymphocytes and mononuclear cells. The present approach is just the opposite of the conventional method of layering blood over a density gradient solution. In the conventional method a required amount of density gradient solution is first placed in a centrifuge tube and then blood or marrow is layered on top using a pasteur pipette.
In the present method a required amount of blood or marrow is first placed in a centrifuge tube. The required amount of density gradient solution is then gently delivered underneath the blood sample using the newly devised density gradient dispenser. Because of the difference in densities, the released density gradient solution lifts the sample of blood upwards and produces exactly the same blood and density gradient suspension as that produced by the conventional method but is less cumbersome, faster and more convenient.
Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality
This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality.
For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups.
For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density–1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension.
OptiPrep? Density Gradient Medium | ||||
| M1248-100 | Biovision | each | 301.2 EUR | |
OptiPrep? Density Gradient Medium | ||||
| M1248-250 | Biovision | each | 405.6 EUR | |
Nycodenz AG ® | ||||
| 1002424 | Axis-Shield Density Gradient | 500g | 1108 EUR | |
8 Regulated Phthalates and BPA in Medium Density Polyethylene | ||||
| CRM-PE002 | Scientific Laboratory Supplies | 5G | 234.84 EUR | |
GRADIENT STOCK SOLUTION | ||||
| 99-674-CM | CORNING | 1 L /pk | 736.8 EUR | |
ISLET GRADIENT 1.037 | ||||
| 99-690-CIS | CORNING | 75 mL/pk | 122.4 EUR | |
ISLET GRADIENT 1.096 | ||||
| 99-691-CIS | CORNING | 75 mL/pk | 160.8 EUR | |
ISLET GRADIENT 1.108 | ||||
| 99-692-CIS | CORNING | 75 mL/pk | 180 EUR | |
ISLET GRADIENT 1.069 | ||||
| 99-815-CIS | CORNING | 75 mL/pk | 141.6 EUR | |
Mastercycler Nexus Gradient | ||||
| E6331000041 | Scientific Laboratory Supplies | EACH | 8391.54 EUR | |
anti-Anterior Gradient 2 | ||||
| YF-PA25612 | Abfrontier | 50 ul | 400.8 EUR | |
Mastercycler nexus gradient eco | ||||
| E6334000042 | Scientific Laboratory Supplies | EACH | 6826.32 EUR | |
Mastercycler nexus GSX1 gradient | ||||
| E6345000044 | Scientific Laboratory Supplies | EACH | 9669.48 EUR | |
A Medium | ||||
| DJ1018 | Bio Basic | 100g | 101.76 EUR | |
DC MEDIUM | ||||
| D04-117-10kg | Alphabiosciences | 10 kg | 1894.8 EUR | |
DC MEDIUM | ||||
| D04-117-2kg | Alphabiosciences | 2kg | 458.4 EUR | |
DC MEDIUM | ||||
| D04-117-500g | Alphabiosciences | 500 g | 168 EUR | |
Advanced Medium | ||||
| C0003-04 | Addexbio | RT 500 mL Bottle | 123.6 EUR | |
With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.