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

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

3. I I I CH 3 COHN NHCOCH 3 COOH Diatrizoic acid (MWt 614) Hypaque  Axis-Shield Density Gradient Media I

4. Nycodenz (MWt 821) CONHCH 2 CHCH 2 OH OH NCH 2 CHCH 2 OH OHCO CH 3 CHCH 2 NHCO CH 2 OH OH II I Axis-Shield Density Gradient Media II

5. CONHCH 2 CHCH 2 OH OH CONHCH 2 CHCH 2 OH OH NCH 2 CHCH 2 N OHCO CH 3 CHCH 2 NHCO CONHCH 2 CH CH 2 OH OH I I II I I Iodixanol (MWt 1550) Axis-Shield Density Gradient Media III

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

7. Concentration (% w/v) Density (g/ml) 0102030405060 1 1.1 1.2 1.3 Sucrose Polysucrose Nycodenz® Iodixanol Density versus concentration

8. Use of density barriers I 1.06 g/ml 1.05 g/ml1.07 g/ml1.10 g/ml

9. 1.06 g/ml 1.05 g/ml1.07 g/ml1.10 g/ml Use of density barriers II (mixer)

10. 1.05 g/ml1.07 g/ml1.10 g/ml 1.07 g/ml 1.06 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. Concentration (% w/v) Density (g/ml) 0102030405060 1 1.1 1.2 1.3 Sucrose Polysucrose Nycodenz® Iodixanol Density versus concentration

13. Sucrose Polysucrose Percoll® Nycodenz® Iodixanol Concentration (% w/v) Osmolality (mOsm) 0102030405060 0 200 400 600 800 1,000 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. 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 Axis-Shield Density Gradient Media

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