Transcript Lipoprotein profiling in self-generated iodixanol gradients

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