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Protein technology topic: TNF-α biological activity detection method
Fundamental
One of the biological activities of TNF is the ability to directly kill tumor cells. When TNF binds to the corresponding receptor and moves into the cell, the lysosomal uptake by the target cell leads to a decrease in lysosomal stability, and various enzymes are leaked, causing cell lysis. The sensitivity of tumor cell lines to TNF-α is very different. Treatment of tumor cells with actinomycin D, mitogen C, and cycloheximide can significantly enhance the activity of TNF-α to kill tumor cells.
Materials and reagents
1, complete medium (1) 10% FCS-DMEM medium (V / V).
2, complete medium (2) 3% FCS-DMEM medium (V / V) 0.5-1μg / ml actinomycin-D.
3,0.05% crystal violet solution:
Take 50 mg of crystal violet, dissolve it with 20 ml of absolute ethanol, dilute to 100 ml with water, and store at room temperature.
4, decolorizing solution:
H 2 O 50mg
Anhydrous ethanol 50ml
Acetic acid 0.1ml
Mix well.
5, TNF standard: provided by the China National Institute for the Control of Pharmaceutical and Biological Products.
Experimental operation
1. This experiment was carried out in a sterile environment. The ultra-clean workbench was irradiated with UV light for more than 30 minutes before the experiment.
2. Adjust the well-preserved mouse L929 cells to a cell suspension of 1×10 5 /ml with complete medium (1), add 100 μg/well to 96-well cell culture plates, 5% CO 2 , and incubate at 37 ° C overnight. .
3. Dilution of standard: The standard was diluted 10 times to 100 IU/ml with complete medium (2), and the plate was diluted 4 times.
4. Dilution of the sample to be tested: The sample to be tested is diluted 10-fold to a certain range with complete medium (2), and then diluted 4 times to prepare the upper plate.
5. Discard the 96-well cell culture plate, add the standard and the sample to be tested to a 96-well cell culture plate at 100 μl/well, and set a control group and a blank group, 5% CO 2 , and culture at 37 ° C for 16 hours.
6. After microscopic examination, discard the supernatant, add 30μl of 0.05% crystal violet to each well for 3~5 minutes, carefully rinse away the crystal violet with running water, dry the residual water in the culture well, add 100μl/well of decolorization solution, use The optical density value at 570 nm was measured by a microplate reader.
Result calculation
1, on the coordinate paper, the OD570 colorimetric value (the average of the two-hole colorimetric value) is plotted against the dilution factor, and the paper with the highest paper and the highest average value of the standard is equal to the X-axis, and the corresponding sample curve is used. At the intersection with the line, the half-effect dilution is read on the X-axis.
2, the calculation formula:
TNF activity (IU/ml) = standard titer x sample pre-dilution factor / standard pre-dilution factor x standard half-effect dilution / sample equivalent to standard half-difference.