Separating Solutions and Lectins

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Biocoll

Biocoll separating solution does contain Ficoll 400® (Polysucrose 400), a polymer with a molecular weight of approx. 400,000 Dalton. Densities of up to 1.1 g/ml can be adjusted using this hydrophilic polymer. For optimal pH and osmolality, adjusting Biocoll with an acid, preferably amidotrizoeic acid (ATA), and sodium hydroxide is required. Biocoll with densities of 1.077 g/ml (cat.no. L 6113/L 6115) and 1.090 g/ml (cat.no. L 6125) are already adjusted accordingly.
Separating lymphocytes using Biocoll

- Biocoll separating solution (density = 1.077 at +20°C) is given to 15 or 25 ml centrifugal tubes in volumes of 7 ml and 10 ml, resp.

- Equal parts of heparinized whole blood (50 IU/ml heparin, stabilizer free) and culture medium are mixed and carefully applied over Biocoll separating solution

- Centrifuge 1200 g for 20 min

- Take layer of enriched (70 - 100%) lymphcytes (between plasma and Biocoll) with a Pasteur's pipette and wash 2x in culture medium:

1. for 10 min at 300 g
2. for 10 min at 200 g

- Cell counting as usual

Pack/Density

Unit

Cat.no.


Biocoll separating solution Density 1.077 g/ml, isoton Storage: 24 months at +2 - +25°C	- / -	100 ml	L 6113

	- / -	500 ml	L 6115



Biocoll separating solution Density 1.09 g/ml, isoton Storage: 24 months at +2 - +25°C	- / -	500 ml	L 6125



Biocoll separating solution Density 1.10 g/ml, isoton Storage: 24 months at +2 - +25°C	- / -	500 ml	L 6155

Mitogens (Lectins)

About 100 years ago, a protein, extracted from Canavalia ensiformis, was found to agglutinate human erythrocytes. The purified protein was named Concanavalin A (Con A). Similar agglutinins from other plant species were identified (80% of them are leguminose species) that agglutinate erythrocytes either species specific or blood group specific. They were named as phytohemagglutinins.

Molecular basis of agglutination:
Phytohemagglutinins do have several binding sites for carbohydrate derivates located in the cell membrane. For instance, Con A readily binds to mannose and glucose. PHA recognizes more complex oligosaccharides. By means of multiple binding, next-neighbour cells are agglutinated.

Molecular basis of mitogen stimulation:
Lymphocytes proved to have an increased mitosis rate under the influence of this phytohemagglutinin. Binding of the mitogen to the cell membrane seems to increase the molecular/information flow between the cell and its environment. Increased membrane permeability activates the synthesis of proteins and nucleic acids. The synthesis rate for messenger substances like lymphokines (e.g. IL-2) is also increased.

Pack/Density

Unit

Cat.no.


Concanavalin A (Con A) lyophilized, for 5 ml Storage: 12 months at +2 - +8°C	- / -	1x (6x5 ml)	M 5050



Phytohemagglutinin (PHA) E lyophilized, for 5 ml Storage: 12 months at +2 - +8°C	- / -	1x (6x5 ml)	M 5040



Phytohemagglutinin (PHA) L lyophilized, for 5 ml Storage: 12 months at +2 - +8°C	- / -	1x (6x5 ml)	M 5030



Pokeweed Mitogen (PWM) Storage: 12 months at +2 - + 8°C	- / -	1x (6x5 ml)	M 5060

Percoll

The basis of Percoll are silica gel particles, coated with PVP (polyvinyl pyrrolidone). BIOCHROM ready-to-use preparations are already adjusted to physiological values of osmolality and pH. Any desired densities from Percoll (cat.no. L 6143/5) can conveniently be prepared with PBS (cat.no. L 1825).
Please contact us for the formula and further details.

Pack/Density

Unit

Cat.no.


Percoll separating solution Density 1.077 g/ml, isoton Storage: 24 months at +2 - +8°C	- / -	100 ml	L 6133

	- / -	500 ml	L 6135



Percoll separating solution Density 1.124 g/ml, isoton Storage: 24 months at +2 - +8°C	- / -	100 ml	L 6143

	- / -	500 ml	L 6145

Separating Solutions and Lectins

Send mail to webmaster with questions or comments about this web site. Copyright © 2005 Gentaur BVBA Last modified: feb-07

Send mail to webmaster with questions or comments about this web site.
Copyright © 2005 Gentaur BVBA
Last modified: feb-07