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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. 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

Purification of cells

Preparation of gradient solutions from general purpose media

axis shield iodinated density gradient media general purpose
Axis-Shield Iodinated Density Gradient Media (General Purpose)
  • Diatrizoate
  • Nycodenz®
  • Iodixanol
axis shield density gradient media i

I

NHCOCH3

CH3COHN

I

I

COOH

Axis-Shield Density Gradient Media I

Diatrizoic acid (MWt 614)

Hypaque

axis shield density gradient media ii

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)

axis shield density gradient media iii

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)

density versus concentration

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
slide8

1.05 g/ml

1.07 g/ml

1.10 g/ml

1.06 g/ml

Use of density barriers I

slide9

1.05 g/ml

1.07 g/ml

1.10 g/ml

1.06 g/ml

Use of density barriers II (mixer)

slide10

1.05 g/ml

1.07 g/ml

1.10 g/ml

1.06 g/ml

1.07 g/ml

Barrier flotation strategy III

axis shield density gradient media for cells
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 concentration1

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
osmolality versus concentration

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
isoosmotic solutions cells c01
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
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
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
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
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
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
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
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
percoll problems iii
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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