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Purification of cells Preparation of gradient solutions from general purpose media Axis-Shield Iodinated Density Gradient Media (General Purpose) • Diatrizoate • Nycodenz® • Iodixanol Axis-Shield Density Gradient Media I I NHCOCH3 CH3COHN I Diatrizoic acid (MWt 614) Hypaque I COOH Axis-Shield Density Gradient Media II CH3 CH2 OH I CHCH2 NHCO CO OH NCH2 CHCH2 OH OH I Nycodenz (MWt 821) I CONHCH2 CHCH2 OH OH Axis-Shield Density Gradient Media III CH3 CH2 OH I CHCH2 NHCO CO CH3 OH CO NCH2 CHCH2 N CH2 OH I CONHCH2 CH OH OH I I I CONHCH2 CHCH2 OH OH Iodixanol (MWt 1550) I CONHCH2 CHCH2 OH OH Axis-Shield Density Gradient Media (General Purpose) • Polysucrose • MWt approx 450,000 Density versus concentration Density (g/ml) 1.3 Nycodenz® Iodixanol 1.2 Polysucrose Sucrose 1.1 1 0 10 20 30 40 Concentration (% w/v) 50 60 Use of density barriers I 1.05 g/ml 1.07 g/ml 1.06 g/ml 1.10 g/ml Use of density barriers II (mixer) 1.05 g/ml 1.07 g/ml 1.06 g/ml 1.10 g/ml Barrier flotation strategy III 1.05 g/ml 1.07 g/ml 1.06 g/ml 1.07 g/ml 1.10 g/ml Axis-Shield Density Gradient Media for Cells • Diatrizoic acid powder • Nycodenz® powder • OptiPrep™: a sterile 60% (w/v) solution of iodixanol in water, density = 1.32 g/ml Principal competition: Percoll® Density versus concentration Density (g/ml) 1.3 Nycodenz® Iodixanol 1.2 Polysucrose Sucrose 1.1 1 0 10 20 30 40 Concentration (% w/v) 50 60 Osmolality versus concentration Osmolality (mOsm) 1,000 800 Nycodenz® 600 Iodixanol 400 Percoll® Polysucrose Sucrose 200 0 0 10 20 30 40 Concentration (% w/v) 50 60 Isoosmotic solutions: cells (C01) • Nycodenz®: Dissolve 27.6 g powder in 3 mM KCl 5mM Tricine/HEPES buffer ( = 1.15 g/ml); make further dilutions with buffered saline, culture medium etc. • Iodixanol: Dilute OptiPrep™ with buffered saline, culture medium etc. • Percoll®: first mix 9 vol. with 1 vol. of 10X saline Axis-Shield Density Gradient Media • Synthesised as X-ray imaging agents • Diatrizoate, Nycodenz® and iodixanol have been clinically tested as X-ray imaging agents Produced under pharmacological conditions: FDA and EU cGMP compliance • Endotoxin: <1 EU/ml (European standard) • Endotoxin: measured levels <0.13 EU/ml What is endotoxin? • Endotoxin is a heat-resistant pyrogen (sometimes called lipopolysaccharide), which is released by Gram-negative bacteria. What are the effects of endotoxin in vivo? • • • • • • A variety of inflammatory responses Hypotension Changes to leukocyte populations Intravascular coagulation Shock Death What are the effects of endotoxin in vitro? • Interacts with CD14 and other receptors • Causes cytokine production in monocytes and macrophages • Activates complement and coagulation cascades • Acts as a B cell mitogen Percoll® problems I • • • • High levels of endotoxin (6-60 EU/ml) Some free polyvinylpyrollidone (PVP) PVP toxic to cells More expensive versions with reduced endotoxin levels or PVP-free Is Percoll® innocuous to cells? Wakefield, J.S. et al (1982) Biochem. J. 202, 795-797 • Peritoneal macrophages, rat liver Kupffer cells, rat testis Leydig cells ingest large numbers of colloidal silica particles at room temperature • Colloidal silica particles adhere to cell surface at all temperatures • Affects adherence of macrophages Percoll® problems: II • Toxicity to Type II lung pneumocytes: Viscardi RM et al (1992) Exp. Lung Res., 18, 225-245 • Poor recoveries of gastric parietal cells; stimulation of acid and cAMP secretion: Benn, SE et al (1987) J. Physiol. 391, 9P • Toxicity to progenitor cells: Mayanagi, T et al (2003) Reproduction, 125, 667-675 • Adherence of silica to erythrocytes causes progressive haemolysis: Kamiyama M. (2002) Personal communication Percoll® problems: III • Poor recovery and function of soil bacteria: Robe, P et al (2003) Eur. J. Soil Biol., 39, 183-190 • Poor viability of plasmodium: Carter, EH et al (1987) Parasitology, 95, 25-30 • Poor yield and purity of intracellular bacteria: Henriquez, V. et al (2003) Appl. Environ. Microbiol., 69, 6268-6271 • Phagocytosed by sea urchin cells: Gross, PS et al (2000) Immunogenetics, 51, 1034-1044