OptiPrep™ is a sterile endotoxin tested solution of 60% iodixanol in water with a density of 1.32 g/ml.

• OptiPrep™ is a ready-made, sterile and endotoxin tested solution with the following specifications:
  Iodixanol: 60% (w/v)
  Density: 1.320 ± 0.001 g/ml
  Osmolality: approx. 170 mOsm
  Endotoxins: < 1.0 EU/ml

• Iodixanol was developed as an X-ray contrast medium and has therefore been subjected to rigorous clinical testing.
• Iodixanol is non-ionic, non-toxic to cells and metabolically inert.
• Iodixanol is approximately a dimer of Nycodenz® and its biological and physicochemical properties are very similar. However, iodixanol solutions have osmolalities approximately half that of Nycodenz® solutions of the same density. Iodixanol solutions can be made isoosmotic at all densities and OptiPrep™ can be added directly to a cell or organelle suspension without changing its osmolality, thus facilitating their purification in a flotation gradient.
• Because of its higher molecular mass, iodixanol forms self-generated gradients more quickly than Nycodenz®. Mammalian and non-mammalian cells purified in iodixanol gradients have high viability.
• Organelles and membrane vesicles are effectively fractionated in either pre-formed or self-generated gradients.
• Viruses purified in iodixanol gradients show an infectivity: particle ratio at least 100x higher than those from CsCl gradients.
• Protein-protein and protein-nucleic acid complexes are better preserved in iodixanol than in sucrose or glycerol gradients.
• Plasma lipoproteins analyzed in self-generated gradients within 4h.
• Each batch of OptiPrep™ is checked for endotoxin by the limulus amebocyte lysate (LAL) test. Our goal is to produce batches with an endotoxin level lower or equal to 0.13 EU/ml. For every batch produced a Certificate of Analysis showing the actual values of density and endotoxin levels is made available at www.axis-shield-density-gradientmedia.com.
• OptiPrep™ sterility is claimed according to Ph.Eur. OptiPrep™ is manufactured, packed and released in compliance with:
  1. Current EU Guide to Good Manufacturing Practice
  2. Fresenius Kabi AS Quality System
  3. Fresenius Kabi AS Manufacturing Licence

Nycodenz® is an off-white powder, freely soluble in water. Solutions up to 80% (v/w) with a density of 1.426 g/ml can be prepared. Nycodenz® was originally developed as an X-ray contrast medium and has therefore been subjected to rigorous clinical testing. Nycodenz® can be used for the isolation of cells, subcellular organelles and membranes, macromolecules and viruses.

• Nycodenz® is non-ionic, non-toxic to cells and metabolically inert.
• Nycodenz® forms true solutions. It is therefore easy to remove the medium from the cells after fractionation.
• Nycodenz® is resistant to bacterial degradation.
• Nycodenz® solutions can be autoclaved.
• Nycodenz® is the trademark name for iohexol, whose systematic name is 5-(N-2,3-dihydroxypropylacetamido) 2,4,6-tri-iodo-N-N'-bis(2,3-dihydroxypropyl)isophthalamide. It has a molecular weight of 821. The chemical properties and stability of Nycodenz® are related to its structure. Its high density derives from the presence of a substituted triiodobenzene ring linked to a number of hydrophilic groups which are responsible for the high water solubility of Nycodenz®. It is a non-ionic derivative of metrizoic acid; the carboxyl group present in metrizoic acid is linked to the amine group of 3-amino-1,2- propanediol. The dihydroxypropylacetamido side chain is responsible for the very low toxicity of Nycodenz® compared to metrizamide. The iodinated aromatic nucleus absorbs strongly in the ultraviolet region of the spectrum with an absorbtion maximum of 244 nm.
• Gradients of Nycodenz® can be generated by:
  • Diffusion of a discontinuous gradient; simple preparation within 45 minutes.
  • Tilted tube rotation (Gradient Master™).
  • Two-chamber gradient maker.
  • Freezing/thawing of a solution of uniform density.
  • Centrifugation (self-generated gradient).
• The density of Nycodenz® in solution can be determined by measuring the refractive index or it can be calculated from absorbance measurements
• Nycodenz® is a non-colloidal medium therefore the distribution of cells in gradients can be determined using a haemocytometer, electronic particle counter or by light scattering measurements using a spectrophotometer.
• Nycodenz® does not interfere with the orcinol and diphenylamine reactions for the estimation of nucleic acids nor with the very sensitive dye binding assays for protein and DNA.
• Polysaccharides and sugars can be determined in the presence of Nycodenz® using the phenol/H2SO4 assay.
• Fluorimetric assays of nucleic acids and proteins can also be carried out in the presence of Nycodenz®.
• Nycodenz® does not interfere with most assays for the marker enzymes of subcellular components, also commercial scintillants are compatible with Nycodenz®.
• Nycodenz® can be removed from samples by dialysis, ultrafiltration or gel filtration.
• Cells, subcellular organelles and other particulate matter can be isolated from Nycodenz ® by centrifugation without the risk of contaminating the pellet with Nycodenz®.
• Nycodenz® is supplied as a powder

A simple and effective method for the isolation of mononuclear cells from human blood was reported by Dr. Arne Bøyum in 1968. For more than 35 years a commercial medium known as Lymphoprep™ has been widely used for isolating these cells. Mononuclear cells (monocytes and lymphocytes) have a lower buoyant density than the erythrocytes and the polymorphonuclear (PMN) leukocytes (granulocytes). The vast majority of mononuclear cells have densities below 1.077 g/ml. These cells can therefore be isolated by centrifugation on an isoosmotic medium with a density of 1.077 g/ml, which allows the erythrocytes and the PMNs to sediment through the medium while retaining the mononuclear cells at the sample/medium interface.
The described method is rapid, simple and reliable and gives excellent results with blood samples from normal individuals and patients. To obtain the maximum yield it is important that the blood sample is diluted 1:1 with physiological saline before being applied to the Lymphoprep™ The contamination of erythrocytes in the mononuclear cell suspension is usually between 3-10% of the total cell number. Some immature PMNs may band with the lymphocytes during intense immunosuppressive therapy. It is essential to remove most of the platelets from mononuclear cell preparations in order to avoid inhibition in the cytotoxicity test.

• Lymphoprep™ has the same specifications as the expensive PLUS or PREMIUM media from other manufacturers. Lymphoprep™ is a ready-made, sterile and endotoxin tested solution with the following specifications:
  • Sodium diatrizoate: 9.1% (w/v)
  • Polysaccharide: 5.7% (w/v)
  • Density: 1.077 ± 0.001 g/ml
  • Osmolality: 290 ± 15 mOsm
  • Endotoxins: < 1.0 EU/ml
    • Each batch of Lymphoprep™ is checked on the level of endotoxins using a specific LAL test. Axis-Shield's goal is to produce batches with an endotoxin level lower or equal to 0.13 EU/ml.
    • Sterility is claimed according to Ph.Eur.
    • Lymphoprep™ is manufactured, packed and released in compliance with:
      • 1. Current EU Guide to Good Manufacturing Practice
      • 2. Fresenius Kabi AS Quality System
      • 3. Fresenius Kabi AS Manufacturing Licence
  • For every lot, a Certificate of Analysis showing the actual values of density, osmolality and endotoxins is made available at www.axis-shield-density-gradient -media.com.
    
• Lymphoprep™ can be used for the preparation of pure lymphocyte suspensions for tissue typing, antilymphocyte sera and immunological research. Thorsby and Brattelie used this technique with only slight modifications in the preparation of pure lymphocyte suspensions for cytotoxicity tests and lymphocyte cultures.

The success of the standard method for isolation mononuclear cells using Lymphoprep™ depends to a large extent on the careful layering of the diluted blood sample on top of the centrifugation medium to maintain a sharp interface between the two layers. This procedure requires some practise and can be time-consuming with large numbers of samples.

Lymphoprep™ Tube is a sterile tube in which the Lymphoprep ™ is contained below a plastic filter disc. This allows blood to be poured simply and directly into the tube, the disc preventing any mixing with the separation medium. The erythrocytes displace the Lymphoprep™ upwards to allow mononuclear cells to band above the filter disc.

Lymphoprep™ Tube can be used for the preparation of pure lymphocyte suspensions for tissue typing, antilymphocyte sera and immunological research. Thorsby and Brattelie used this technique with only slight modifications in the preparation of pure lymphocyte suspensions for cytotoxicity tests and lymphocyte cultures.

Lymphoprep™ Tube is manufactured to the same rigourous specifications as given above for Lymphoprep™.