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Purification of cells

Purification of cells. Preparation of gradient solutions from general purpose media. Axis-Shield Iodinated Density Gradient Media (General Purpose) . Diatrizoate Nycodenz ® Iodixanol. I. NHCOCH 3. CH 3 COHN. I. I. COOH. Axis-Shield Density Gradient Media I. Diatrizoic acid (MWt 614)

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Purification of cells

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  1. Purification of cells Preparation of gradient solutions from general purpose media

  2. Axis-Shield Iodinated Density Gradient Media (General Purpose) • Diatrizoate • Nycodenz® • Iodixanol

  3. I NHCOCH3 CH3COHN I I COOH Axis-Shield Density Gradient Media I Diatrizoic acid (MWt 614) Hypaque

  4. CH3 CO OH CH2 OH I NCH2 CHCH2 OH CHCH2 NHCO OH I I CONHCH2 CHCH2 OH OH Axis-Shield Density Gradient Media II Nycodenz (MWt 821)

  5. CH3 CH3 CH2 OH CH2 OH I CO OH CO I CHCH2 NHCO CONHCH2 CH NCH2 CHCH2 N OH OH I I I I CONHCH2 CHCH2 OH CONHCH2 CHCH2 OH OH OH Axis-Shield Density Gradient Media III Iodixanol (MWt 1550)

  6. Axis-Shield Density Gradient Media (General Purpose) • Polysucrose • MWt approx 450,000

  7. Density (g/ml) 1.3 Nycodenz® Iodixanol 1.2 Polysucrose 1.1 Sucrose 1 0 10 20 30 40 50 60 Concentration (% w/v) Density versus concentration

  8. 1.05 g/ml 1.07 g/ml 1.10 g/ml 1.06 g/ml Use of density barriers I

  9. 1.05 g/ml 1.07 g/ml 1.10 g/ml 1.06 g/ml Use of density barriers II (mixer)

  10. 1.05 g/ml 1.07 g/ml 1.10 g/ml 1.06 g/ml 1.07 g/ml Barrier flotation strategy III

  11. 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®

  12. Density (g/ml) 1.3 Nycodenz® Iodixanol 1.2 Polysucrose 1.1 Sucrose 1 0 10 20 30 40 50 60 Concentration (% w/v) Density versus concentration

  13. Osmolality (mOsm) 1,000 800 Nycodenz® 600 Iodixanol 400 Percoll® Polysucrose Sucrose 200 0 0 10 20 30 40 50 60 Concentration (% w/v) Osmolality versus concentration

  14. 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

  15. 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

  16. What is endotoxin? • Endotoxin is a heat-resistant pyrogen (sometimes called lipopolysaccharide), which is released by Gram-negative bacteria.

  17. What are the effects of endotoxin in vivo? • A variety of inflammatory responses • Hypotension • Changes to leukocyte populations • Intravascular coagulation • Shock • Death

  18. 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

  19. 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

  20. 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

  21. 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; stimul-ation 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

  22. 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

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