Determination of the biological activity of lecytosin-2 (IL-2).
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Last Update: 2020-10-27
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Source: Internet
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Author: User
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the purpose of the experiment
. 1. Master the principles of THEL-2 biological activity determination.
2.Be familiar with the experimental methods and results of IL-2 biological activity determination.
principle of experimentation
. IL-2 is a lymph factor produced by Th cells, which plays a very important role in the process of lymphocyte proliferation and differentiation. IL-2 activity determination is based on IL-2 to maintain the metabolism and survival of IL-2 dependent cells and promote the proliferation of such cells. Cells are active in energy metabolism during proliferation.
produces a large amount of energy for synthesis of a variety of large molecular substances and cell division, the level of energy metabolism and cell
. DNA
levels are basically parallel, so measuring the level of cellular energy metabolism indirectly reflects cell proliferation.
MTT (dimethyl azoazine) is a pale yellow water-soluble compound in which living cells (especially those during the proliferation period) are caused by the role of amber acid
dehydrogenase
in mitochondrial energy metabolism. TT decomposes to produce blue crystalline armordeposited in or around cells, and the amount of A formed is directly related to the degree of cell proliferation, A after the
SDS
action can be dissolved color. The light density value of the solution is positively related to cell metabolism and IL-2 activity.
the experimental material
. 1. 1640
cultured
liquid (full), IL-2
standard
, IL-2 sample to be tested, CTLL-2 cell strain, MTT solution (5mg/ml), 10% SDS
2.96-
cell culture
plate, multi-headed cell collector, microscope, CO2 incubator, enzyme marker
experimental method
1.Prepare CTLL-2 cell suspension to take the vigorously growing CTLL-2 cells, wash the cells 3 times with 1640 culture fluid, and match the cell suspension with a complete 1640 culture solution to 2×105/ml cell suspension.
2.Diluted samples and standards The samples to be tested and the standard IL-2 are diluted by a certain ratio with a complete 1640 culture solution.
3.Samples and standards (50 μl/hole) with different dilutions were added to the 96-well cell culture plate, with 3 compound holes per dilution and cell control. Then add 50 sl cell suspension to each hole, mix well, set 5% CO237 degrees C culture for 36 hours, add MTT solution 20 μl CO2 incubation 6 to 8 hours, add 10% SDS100 sl per hole, mix well, 37 degrees C incubator static (make a meth fully dissolved).
the wavelength 570nm on the enzymatic instrument to determine the OD value, the OD value of the sample to be tested with the standard OD value, to find the IL-2 active unit of the sample to be tested.
results are calculated
the OD values of each dilution degree are converted into probability units as a percentage of the sample's maximum proliferation OD value, and the original S-shaped curve can be converted into a straight line. The regression equations for each straight line are based on the data of these points. Then from the regression equation to find each sample up to 50% maximum proliferation dilution, and then according to the formula to find the active unit of the sample to be tested IL-2.
notes are
. 1. MTT liquid should be ready to use, avoid lighting, if there are blue particles need to
filter
before use.
2. Biological assays are highly sensitive and specific, and the conditions and requirements of the measurements are strictly in accordance with procedures, as is the case with the bioactive IL-2, rather than the immunoreactive IL-2 proteins measured by
immunology
assays.
.
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