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.

OptiPrep? Density Gradient Medium

M1248-100 Biovision 251 EUR

OptiPrep? Density Gradient Medium

M1248-250 Biovision 338 EUR

GRADIENT STOCK SOLUTION

99-674-CM CORNING 1 L /pk 614 EUR

ISLET GRADIENT 1.037

99-690-CIS CORNING 75 mL/pk 102 EUR

ISLET GRADIENT 1.096

99-691-CIS CORNING 75 mL/pk 134 EUR

ISLET GRADIENT 1.108

99-692-CIS CORNING 75 mL/pk 150 EUR

ISLET GRADIENT 1.069

99-815-CIS CORNING 75 mL/pk 118 EUR

anti-Anterior Gradient 2

YF-PA25612 Abfrontier 50 ul 334 EUR

M9CA Medium

SD7025 Bio Basic 250g 71.75 EUR

YM Medium

SD7031 Bio Basic 250g 70.45 EUR

TYGPN Medium

SD7032 Bio Basic 250g 70.01 EUR

M63 Medium

SD7033 Bio Basic 250g 71.75 EUR

M9 Medium

SD7024 Bio Basic 250g 71.75 EUR

A Medium

DJ1018 Bio Basic 100g 84.8 EUR

NeuroProgenitor Medium

NM42400 Neuromics 125 ml 304 EUR

Heller's Medium

CP014-010 GenDepot 10X1L 99 EUR

Heller's Medium

CP014-500 GenDepot 50L 126 EUR

Hoagland's Medium

CP015-010 GenDepot 10X1L 99 EUR