Mouse tnf α quantikine elisa kit mta00b

Levels of cytokines, including IL-1β and TNF-α, both in culture supernatants and in serum, were measured by using the mouse IL-1β/IL-1F2 Quantikine ELISA Kit (MLB00C; R&D Systems, Inc., Minneapolis, MN, USA) and mouse TNF-α Quantikine ELISA Kit (MTA00B; R&D Systems, Inc.), respectively, according to the manufacturer's instructions. Absorbance at 490 nm was then measured with an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).

Raw 264.7 cells (100,000) were grown in 96-well plates. After 24 h, cells were stimulated with vehicle (final concentration of 0.025 % dimethyl sulfoxide (DMSO)) or with apremilast at the indicated concentrations. After 30 minutes cells were stimulated with LPS (L5886; Sigma, St Louis, MO, USA) 1 μg/ml for 4 h. When studying CGS21680 (1063; Tocris Bioscience, Ellisville, MO, USA), SCH58261 (2270; Tocris Bioscience), ZM241385 (1036; Tocris Bioscience), BAY60-6583 (4472; Tocris Bioscience), or GS6201 (4727; Tocris Bioscience), the adenosine receptor ligands were added 15 minutes before apremilast. Methotrexate (Hospira Inc, Lake Forest, IL, USA) was added 24 h and 1 h before apremilast. Supernates were then collected and TNF-α levels were quantified with the Mouse TNF-α Quantikine ELISA Kit (MTA00B; R&D systems; Minneapolis, MN, USA) following the manufacturer’s instructions.