Mouse tnfα opteia elisa kit

Bone marrow single cell suspension was obtained from femurs of MV130 or excipient pretreated mice, as described above. Afterward, cells were cultured in R10, counted and plated at equal number (10⁵ cells/well) in 96-well flat bottom wells. Following an already published protocol (Christ et al., 2018 (link)) cells were incubate for 6 h with LPS (10ng/ml; TLR4 ligand) or with R10 as control and TNF-α levels were subsequently measured in the supernatants by ELISA (Mouse TNFα Opteia ELISA kit, BD Biosciences).

MV130 and excipient pretreated mice were sacrificed in a CO₂ chamber to collect femurs and tibia that were subsequently flushed with cold PBS to harvest bone marrow. RBC lysis was performed once for 3 min at room temperature (RT). To obtain BMDM, following an already published paper (Saz-Leal et al., 2018 (link)), cell suspensions were plated in non-treated cell culture plates (Corning) in R10, plus M-CSF (30% mycoplasma-free L929 cell supernatant) at 37°C for 5 days. At day 5, BMDM were detached in phosphate buffered saline (PBS, Gibco) supplemented with 5 mM EDTA (PBS/EDTA, Life Technologies), counted and subsequently plated in R10, at equal number (10⁵ cells per well) in 96-well plates (200-μL final volume, Corning) and rested overnight. Then, cells were primed with 25 ng/mL IFNγ (BD Biosciences, San Jose, CA) for 24h. After that, cells were washed and stimulated with R10 or 1 μg/mL check Escherichia coli LPS (EK, Invivogen, San Diego, CA) overnight. Supernatants were subsequently collected for TNFα measurement by ELISA (Mouse TNFα Opteia ELISA kit, BD Biosciences).