Density Gradient Media - News and Views

Lipid raft fractionation using OptiPrep

JohnG — Thu, 20 May 2010 12:10:08 +0000

Lipid-rich plasma membrane domains have commonly been isolated from tissues and cultured cells as detergent-resistant membranes (DRMs), principally by flotation from a dense solution through a discontinuous or continuous density gradient containing a non-ionic detergent, usually Triton X-100 (TX100), but sometimes CHAPS. The resolution of DRMs is based on their low buoyant density and a flotation strategy is regarded as the best method for resolving any minor low-density fraction from predominantly denser material. Sucrose gradients were originally used in the separations but the much lower viscosity of Nycodenz solutions, compared to those of sucrose, allowed the centrifugation time at 200,000 g to be reduced from 18 h to 4 h. Although iodixanol gradients are slightly more viscous than the corresponding Nycodenz ones, the ease of preparation of gradient solutions from a commercial 60% (w/v) solution (OptiPrep) has made use of this medium very popular.

An alternative approach, which avoids the use of a detergent, also uses an iodixanol gradient to resolve the low-density lipid-rich plasma membrane domains.

Click HERE to access an Axis-Shield Mini-review of the detergent-based methods. It contains a brief methodological survey of the technique and a bibliography of all of the current papers (up to late 2009) that have reported the use of an iodixanol gradient to purify and analyze the lipid rafts.

Click HERE to access a detailed protocol of the detergent-based method.

Click HERE to access a detailed protocol of the detergent-free method.

Isolation of human monocytes using OptiPrep

JohnG — Tue, 18 May 2010 14:52:44 +0000

Flotation of monocytes from a leukocyte-rich fraction through a simple discontinuous iodixanol gradient provides a simple method for their purification. Both Graziani-Bowering et al [1] and later Nutt et al [2] found that the purity based on CD14/CD4 [1] or CD68 [2] analysis was at least 90%. Nutt et al [2] compared the monocyte isolates with those from centrifugal elutriation and concluded that they were comparable. The method is easy, rapid and avoids the use of special expensive rotor hardware. More recently [3] it has been recommended in studies on monocyte-derived macrophages.

Yields by this flotation method, which is carried out at room temperature, are approx. 35%. Recently an alternative even more rapid flotation method was developed. It uses whole blood and is carried out at a lower temperature; the yields are raised to approx 60%, but the purity is reduced to approx. 80%.

Detailed protocols of both monocyte isolation strategies, from a leukocyte-rich plasma (Application Sheet C09) and from whole blood (Application Sheet C10) and abstracts of papers reporting the use of these methods may be accessed from the Cell Index on the Axis-Shield website; click HERE and follow the instructions.

1 Graziani-Bowering, G.M., Graham, J. and Filion, L.G. (1997) A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes J. Immunol. Methods, 207, 157-168

2 Nutt, J.C., Willis, C.C., Morris, J.M. and Gallery, E.D.M. (2004) Isolating pure populations of monocytes from the blood of pregnant women: comparison of flotation in iodixanol with elutriation J. Immunol. Methods, 293, 215-218

3 Quesniaux, V., Erard, F. and Ryffel, B. (2010) Adjuvant activity on murine and human macrophages In Vaccine Adjuvants (ed. Davies, G.) Methods Mol. Biol., 626, 117-130, Humana Press, Totowa, NJ, USA

Purification of neurons for cell culture

JohnG — Wed, 12 May 2010 13:18:51 +0000

The purification in Nycodenz gradients and subsequent culture of neurons from adult rat hippocampus was described by Brewer [1] in 1997. Four years later Brewer et al [2] adapted the method to human cortical brain tissue, taken at craniotomy and using iodixanol gradients. Of all the gradient media currently available commercially, iodixanol is probably the most “cell-friendly” and its availability as a sterile 60% (w/v) solution (OptiPrep) makes gradient solution preparation simple. Use of an iodixanol gradient improves both the yield and viability of neurons [3], important factors in the successful establishment of neuron cultures.

Iodixanol density gradients are now widely used for the purification of neurons from human, hamster, mouse, rat and turtle brain tissues and from chicken, rat and mouse spinal cord.

1. Brewer G.J. (1997) Isolation and culture of adult rat hippocampal neurons J. Neurosci. Methods 1997: 71: 143-155

2. Brewer, G.J. et al (2001) Culture and regeneration of human neurons after brain surgery J. Neurosci Methods, 107, 15-23

3. Brewer, G.J. and Torricelli, J.R. (2007) Isolation and culture of adult neurons and neurospheres Nat. Protoc., 2, 1490-1498

For a complete bibliography click on Mini-review

For a detailed methodology click on brain or spinal cord

Purification of rAAV for CNS transduction

JohnG — Mon, 19 Apr 2010 09:06:17 +0000

Compared to CsCl and sucrose there are considerable procedural advantages to the use of OptiPrep for the purification of rAAV.

OptiPrep is a sterile solution of 60% (w/v) iodixanol; simply dilute it with saline to prepare sterile gradient solutions. It is the only gradient medium manufactured under strict FDA and EU cGMP compliance.

CsCl and sucrose are toxic to cells and must be removed prior to infection of cells. Iodixanol is non-toxic to cells; it has very low endotoxin levels; measured levels on each batch are <0.13 EU/ml. No dialysis is required before re-infection of cells. A Certificate of Analysis is available for each batch.

CsCl gradients lead to major reductions in infectivity of rAAV: higher recovery of rAAV from iodixanol gradients compared to CsCl gradients and average particle number /infectivity ratios much lower in iodixanol isolates compared to CsCl isolates

CsCl must be removed prior to HPLC or gel electrophoresis; iodixanol rarely needs removing prior to further processing.

For more information on the purification of rAAV and an up-to-date reference list click here.

Endocytosis analysis

JohnG — Mon, 12 Apr 2010 09:39:45 +0000

Axis-Shield’s density gradient media – Nycodenz and OptiPrep can provide higher resolution of endosomal compartments than the traditional sucrose gradients. The new Axis-Shield Mini-Review No. 3 [2010] provides a summary of the technology as applied to both tissues and cultured cells. In addition to the routine pre-formed gradients, OptiPrep can also offer a self-generated mode that makes gradient formation simple and highly reproducible.

A companion Mini-Review – No. 2 [2010] – provides a reference list of the all the known papers reporting the use of both Nycodenz and OptiPrep in endocytosis analysis. The list is divided into tissue and cell type and, when merited by the number of references, also into functional study.

Detailed methodologies are given in a series of Axis-Shield Application Sheets that are listed in Mini-Review 2 [2010]. Electronic links to these Application Sheets are provided in Mini-Review 2 [2010].

Cell culture from biopsy tissue

JohnG — Fri, 09 Apr 2010 09:15:49 +0000

One of the focuses of tissue engineering and regenerative medicine research is the efficient in vitro growth of cells from biopsy material on a plastic substratum, as a prelude to its eventual use clinically. A recent paper by Bruce et al [1] reported on the culture of urothelial cells from non-neurogenic and neurogenic human bladder tissue. The publication described the suspension of the cells in a 12% (w/v) iodixanol solution, overlaid with a small layer of culture medium. During centrifugation this allowed the viable epithelial cells to float to the surface while non viable cells, debris and erythrocytes sediment. The absence of an interface at which viable cells and non-viable cells might aggregate together is a critical feature of this system. The authors reported that the iodixanol “clean-up” increased the percentage of attached cells 24 h after plating from 27% to 84% and that multiple large colonies of cells were observed with the purified cells.

¨ The well-established low endotoxin levels in OptiPrep and the clinical acceptability of iodixanol as an X-ray imaging agent underline the importance of applications in tissue engineering and regenerative medicine research

Bruce, A.T., Sangha, N., Richmond, A., Johnson, K., Jones, S., Spencer, T. and Ludlow, J.W. (2010) Use of iodixanol self-generated density gradients to enrich for viable urothelial cells from non-neurogenic and neurogenic bladder tissue Tissue Eng., Part C Methods 16, 33-40

Axis-Shield Application Sheet C13 provides detailed methodological options for viable cell enrichment and can be accessed by clicking here.

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admin — Tue, 09 Feb 2010 14:52:55 +0000

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