A sensitive new bioassay for tumor necrosis factor.
Shahan-TA; Siegel-PD; Sorenson-WG; Kuschner-WG; Lewis-DM
J Immunol Meth 1994 Oct; 175(2):181-187
An improved tumor necrosis factor (TNF) bioassay using an isolated subclone from the murine fibroblastoid NCTC-clone-929 cell line and a new fluorescence indicator system for detecting target cell viability was described. The NCTC-clone-929 cell line was cultured and cloned. Target cells were released from attachment, centrifuged, counted, and diluted to a density of 2x10(4) cells per well before a 24 hour incubation with TNF-alpha standards or unknowns. After removal of medium and sample, the cells were incubated with 10% Alamar-blue for 12 hours. Alamar-blue fluorescence was read on a fluorescence 96 well plate counter at 560 to 590 nanometers. Neutral-red and crystal-violet assays were performed for comparison with the Alamar-blue bioassay. Cells harvested from CD-rats by bronchoalveolar lavage were isolated and then incubated with varying concentrations of lipopolysaccharide (LPS) for the detection of TNF activity in LPS macrophage conditioned medium. A TNF hypersensitive subclone A12 from the NCTC- clone-929 was isolated and found to be sensitive to as little as 68+/-2.5 femtograms/milliliter of TNF-alpha. Incubation of the cells in different sera and varying serum concentrations did not indicate any significant differences between sera, but revealed a significant reduction in sensitivity if less than 15% serum was used. A comparison of crystal-violet and neutral-red as indicators of viability showed that the Alamar-blue bioassay was three and four times more sensitive, respectively. TNF activity was detected with 3.0 nanograms/milliliter LPS. The authors conclude that the Alamar- blue assay employing the NCTC-clone-929 subclone A12 is effective in the quantitation of low levels of TNF, particularly when a suitable immunoassay is unavailable.
NIOSH-Author; Cell-cultures; Cytochemistry; Bioassays; Mammalian-cells; Biochemical-indicators; Immunochemistry; Cell-culture-techniques
Journal of Immunological Methods