Cytokines and nitrite (an indirect indicator of NO production) were measured in cell culture supernatants from BMM and PuM un-treated or treated with either IFN-γ or AECsup at 4, 24 and 48 h after the end of infection with BCG. For ELISA determinations, the commercially, TNF, IL-6, CXCL10/Interferon gamma-induced protein 10 kDa (IP-10), IL-1β (R&D Systems) and IL-12, (Mabtech) were used to determine the cytokine levels in the culture supernatants according to the manufacturer's recommendations. The enzyme-substrate reaction was developed using p-nitrophenyl phosphate (Sigma) for IL-12 and tetramethylbenzidine substrate (R&D Systems) for the rest of determinations. Depending on the substrate used, the optical density was measured in a multiscan ELISA reader (Anthos Labtech Instruments, Salzburg, Austria) at 405 or 450 nm. NO production was determined by measuring nitrite concentration using the Griess reaction according to the manufacturers protocol (Sigma).
Il 12
Manufactured by Mabtech
Sourced in Sweden
IL-12 is a cytokine that plays a crucial role in the regulation of the immune system. It is involved in the differentiation and expansion of T-helper cells and natural killer cells, and it enhances the cytotoxic activity of these cells. IL-12 is commonly used in immunological research and assays to study various aspects of cellular immune responses.
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2 protocols using il 12
1
Cytokine and Nitrite Response to BCG
2
Quantifying IL-12 in Bone Marrow Macrophages
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The level of IL-12 produced in BMMs with TLA (100 µg/100 µl) treatment for 24 hr was measured using the sandwich ELISA method. The capture antibody (IL-12) (Mabtech Inc., Nacka Strand, Sweden) was diluted to 2 µg/ml in PBS (pH 7.4), coated on a 96-well plate (Costar, New York, USA) at 100 µl/well, and incubated overnight at 4℃. After the blocking step, the culture supernatant was added to the wells, and incubated for 2 hr. Then, a biotin-conjugated anti-IL-12 antibody (Mabtech Inc.) was applied and incubated for 1 hr. Finally, streptavidin-HRP (Mabtech Inc.) was added and incubated for 1 hr. TMB solution was used for the enzymatic reaction. OD levels at 450 nm were measured using a spectrophotometer (Molecular Device, Menlo Park, California, USA).
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