Tumour necrosis factors
10.18 Tumour necrosis factors
Tumour necrosis factors α (TNF-α) and β (TNF-β—sometimes referred to as lymphotoxin) are dis- tinct and separate gene products but have similar functions and activities in vitro, and appear to bind to the same receptor.
TNF-α was originally described as a monocyte/macrophage product derived from certain macrophage-like cell lines, but also produced by T cells. It has cytostatic and cytocidal effects on transformed cells; it stimulates bone resorption and inhibits bone reformation; and it inhibits lipoprotein lipase activity in adipocytes.
TNF-β is produced by T lymphocytes and has properties similar to TNF-α. The only effective means of distinguishing between the two molecules at present is by the use of appropriate neutralizing antibodies. Identical assays can be used for both TNF-α and TNF-β.
10.18TUMOUR NECROSIS FACTORS
10.18.1 L929 cell-killing assay
MATERIALS AND EQUIPMENT L929 tumour cells Tissue culture medium Fetal bovine serum (FBS) Actinomycin D 96-well microtitre plates Phosphate-buffered saline (PBS) Crystal violet, 1% w/v aqueous solution ELISA reader
Preparation in advance
1 Actinomycin D sensitizes cells for lysis by TNF. L929 cells can vary in their susceptibility to TNF. It is advisable to work with cell cultures maintained and subcultured for relatively short periods. If TNF assays are to be done on a regular basis, it is recommended that a large number of cells with proven sensitivity to TNF be cryopreserved. Frozen vials can then be used to start up fresh cultures at regular intervals.
2 Subculture the cell line 24 h prior to use to ensure that the cultures are subconfluent.
METHOD
1 Prepare a suspension of L929 cells, by trypsin treatment of a subconfluent culture, and wash twice by centrifugation.
2 Resuspend the cells at 4 × 10 5 /ml in medium containing 5% serum.
3 Dispense 100 µl aliquots of cells into individual wells of microtitre plates.
4 Incubate for 4 h at 37°C in a humidified atmosphere of 5% CO 2 in air to allow the cells to adhere.
5 Remove the medium by inverting the plate with a rapid flicking motion.
6 Add 100 µl aliquots of a range of dilutions of the samples under test, in medium containing 2% serum and actinomycin D (2 µg/ml initial concentration).
7 Incubate the plates overnight at 37°C in a humidified atmosphere of 5% CO 2 in air.
8 Remove the medium as described and wash the wells once with PBS.
9 Remove the PBS by flicking the plate as described and add 100 µl methanol to each well to fix the cells.
10 Remove excess methanol and dry the plates briefly in air.
11 Add 100 µl of aqueous crystal violet and incubate at room temperature for 5 min.
12 Wash the wells thoroughly with tap water; finally empty the wells by flicking the plate.
13 Solubilize the contents of the wells with 100 µl of 33% acetic acid (in water) and read the plates on an ELISA reader at a test wavelength of 570 nm and a reference wavelength of 420 nm.
Assessment of results Reduced OD relative to controls without TNF indicates cell killing. Plot OD against sample dilu-
tion to determine the dilution of the unknown sample that gives 50% target-cell death.
C H A P T E R 1 0: The cytokines
10.18.2 WEHI-164 killing assay
The recently derived subclone 13 of the WEHI-164 murine fibrosarcoma has provided a highly sensitive assay for TNF/LT (lymphotoxin) activity; it is similar in principle to the standard L929 but has the following modifications.
1 Actinomycin D is not required in the medium.
2 The cells are plated at 2 × 10 4 /ml in the presence of the test samples.
3 The culture plates are incubated at 37°C for 20 h. Viability can then be assessed using crystal violet staining (see Section 10.19.1) or the MTT assay (Section 9.5.2).
Assessment of results Reduced OD relative to controls without TNF indicates cell killing. Plot OD against sample dilu-
tion to determine the dilution of the unknown sample that gives 50% target-cell death.