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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 Purpose)

NHCOCH3

CH3COHN

I

I

COOH

Axis-Shield Density Gradient Media I

Diatrizoic acid (MWt 614)

Hypaque


Axis shield density gradient media ii

CH Purpose) 3

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

CH Purpose) 3

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)


Axis shield density gradient media general purpose
Axis-Shield Density Gradient Media (General Purpose) Purpose)

  • Polysucrose

  • MWt approx 450,000


Density versus concentration

Density (g/ml) Purpose)

1.3

Nycodenz®

Iodixanol

1.2

Polysucrose

1.1

Sucrose

1

0

10

20

30

40

50

60

Concentration (% w/v)

Density versus concentration


Purification of cells

1.05 g/ml Purpose)

1.07 g/ml

1.10 g/ml

1.06 g/ml

Use of density barriers I


Purification of cells

1.05 g/ml Purpose)

1.07 g/ml

1.10 g/ml

1.06 g/ml

Use of density barriers II (mixer)


Purification of cells

1.05 g/ml Purpose)

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 Purpose)

  • 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) Purpose)

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 Purpose) )

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) Purpose)

  • 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 Purpose)

  • 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? Purpose)

  • 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 Purpose) 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 Purpose) 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 Purpose) ® 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 Purpose) ® 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 Purpose) ® 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 Purpose) ® 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