Mouse tnf alpha duoset

For mice, TNF levels were measured by ELISA (Mouse TNF-alpha DuoSet, R&D Systems) following manufacturer instructions and normalized to sham stimulated animals. The limit of detection was 15 pg./ml. Porcine samples were analysed by ELISA for quantification of TNF using Porcine DuoSet ELISA kit (Bio-Techne Ltd., cat. no. DY690B). Plates were analysed using the Infinite^® 200 PRO spectrophotometer and iControl software (Tecan Group Ltd.).
Human samples were analysed for concentration of TNF using TNF alpha Human ELISA Kit (Invitrogen, cat. no. KHC3011) and for Ach using the Universal Acetylcholine ELISA Kit (Colorimetric) (Bio-Techne Ltd., cat. no. NBP2-66389) according to manufacturer’s instructions. Plates were analysed using the BMG FLUOstar Optima plate reader (BMG Labtech).

Seven days following implantation, animals were injected i.p. with a lethal dose of lipopolysaccharide (LPS; 400 µg). Electrostimulation was applied using a PlexStim V2.3 (Plexon) starting at − 10, 0 and + 20 min relative to LPS injection. Mice were electrically stimulated with the same parameters as above at 16, 20, 24, 30, 34 and 38 h after LPS injection. Survival was followed over 4 days. Serum was collected at 90 min after LPS injection and assessed for Tumor Necrosis Factor (TNF)-α levels. Again, controls were fully Cortec implanted mice, which did not receive electrical stimulation (sham). Electrostimulation were rectangular charged-balanced biphasic pulses (650 μAmp, 10 Hz, 100 μs per phase) for 2 min. For TNF-α, retro-orbital blood sampling was performed under isoflurane anesthesia. TNF-α levels were measured by ELISA (Mouse TNF-alpha DuoSet, R&D Systems) following manufacturer instructions.

The release of inflammatory factors from the MC3T3‐E subclone 14 cells, including TNF‐α, IL‐1β, and IL‐6, was evaluated using commercially available ELISA kits (Mouse IL‐1 beta/IL‐1F2 DuoSet, Mouse IL‐6 DuoSet, Mouse TNF‐alpha DuoSet, R&D Systems, USA). The cells were cultured in the CO₂ incubator with four different concentrations of ceramic debris (0, 5, 10, and 15 μg/mL) in the culture medium for 3 days, and the concentrations of inflammatory factors in the culture medium were measured based on the manufacturer's instructions for the ELISA kits.