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M5060-BC Pokeweed Mitogen (PWM)

M5060-BC
  
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Áttekintés

Replacement Information
Description
OverviewVegetable lectins are particularly suitable for the stimulation of lymphocytes under in vitro conditions. An important mitogen group is the lectins, sugar-binding proteins. Lectins specifically bind to membrane-glycoproteins of different cells, including lymphocytes. They effect thereby agglutinations producing cell activation and proliferation. Vegetable agglutinins (mostly legumes) can party agglutinate erythrocytes according to the species and blood groups (therefore named phytohemagglutinins or lectins). This includes also Concanavalin A (Con A), a protein extracted from Canavalia ensiformis, which was found to effectively agglutinate human erythrocytes. Phytohemagglutinins (PHA) that mainly agglutinate erythrocytes are referred to as E, whereas PHA with lymphocytes stimulating properties as PHA L. Supplied as 6 x 5 ml.
Specifications:

Functional Test: Pass
pH: 7.33-7.93
Osmolality: 0.043-0.064 Osm/kg H20
Agglutination Titre: 0.11-6.25 µg/ml
Water Content: 0.5-5.0
Color: Colorless
Appearance: Clear
Recommendations:

Following re-constitution in 5 ml a. dest., apply 1 ml per 100 ml medium.

Molecular basis of agglutination:
Phytohemagglutinins do not have several binding sites for carbohydrate derivatives 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 mitogenic 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.

Nomenclature:
Earlier nomenclature of phytohemagglutinines mostly referred to their purification level: PHA M, e.g., was named after residual content of mucoprotein, whereas PHA P indicated the pure protein. The recent nomenclature, however, rather refers to biological characteristics: PHA P preferrably agglutinizes erythrocytes, and is therefore now termed PHA E. PHA M was similarly re-named to PHA L, due to its particularly stimulating effect on lymphocytes.

A routine test of the mitogen stimulation:
Vegetable lectins are perfectly qualified for the stimulation of lymphocytes under in vitro-conditions. The activity of the single lectins here offered is regularly tested on peripheral human lymphocytes. Hereby a short stimulation (72 - 80 h) of the lymphocytes is effected by the respective lectin.
The resulting blast transformation and the mitosis rate are quantitatively determined by the integration of radioactive marked DNA components (mostly 3H-thymidin). To identify the optimal stimulation data the following test system is applied:

• Separation of humane lymphocytes by means of Biocoll separating centrifugation
• 1 x 105 lymphocytes/ml test run (0.2 ml in micro test plates)
• Culture medium: RPMI 1640 with 20 mM HEPES + 10% FBS, pre-tested FBS
• Lectin: adding 10 µl/ml test run
• Radioactive marking: 0.2 µCi 3H-thymidin per test run (spec. act. 2 Ci/mM
• General culture time: 72 - 80 h at +37°C
The processing and measuring of the radioactive marked lymphocytes are carried out by common methods. The substance quantities of the single lectins are set according to the results for maximum stimulation.
Catalogue NumberM5060-BC
Brand Family Calbiochem®
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Ship Code Blue Ice Only
Storage +2°C to +8°C
Do not freeze Ok to freeze
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Specifications
Global Trade Item Number
Katalógusszám GTIN
M5060-BC 0

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