Tnf tn3

Bone marrow cells were derived from the femurs of WT mice and cultured in complete culture medium (RPMI-1640 medium from Gibco, Thermo Fisher Scientific, supplemented with 10% FCS from Gibco, Thermo Fisher Scientific; 1% l-glutamine from MilliporeSigma; and 50 μg/mL gentamicin from Cellgro) and 10 ng/mL GM-CSF (PeproTech). Media were added on day 3. This protocol generates both macrophage and dendritic cell populations (90 (link), 91 (link)). On day 6, cultured cells were collected. The indicated number (10 × 10⁶, 50 × 10⁶, 150 × 10⁶) of cells were transferred i.p. to the recipient mouse 2 hours before LPS challenge (92 (link), 93 (link)). IVCCA (43 (link), 88 (link), 89 (link), 94 (link)–96 (link)) was used to quantify systemic IL-6 and TNF levels. Briefly, biotinylated capture antibodies IL-6 (MP5-32C11) and TNF (TN3) (eBioscience, Thermo Fisher Scientific) were injected i.p. 3 hours before TLR ligand challenge (75 μg ultrapure LPS), and serum cytokine levels were determined 4 hours later.

In vivo, systemic IL-6 and TNF levels were quantified using IVCCA (43 (link), 88 (link), 89 (link), 94 (link)–96 (link)). Briefly, biotinylated capture antibodies IL-6 (MP5-32C11) and TNF (TN3) (eBioscience, Thermo Fisher Scientific) were injected i.p. 3 hours before TLR ligand challenge (25 μg LPS), and serum cytokine levels were determined 4 hours later.
For comparison of maternal serum and amniotic fluid levels of IL-6, samples were collected 6 hours following i.p. (75 μg LPS) or i.a. (5 μg LPS total, administered as 2 × 2.5 μg injections) challenge. Amniotic fluid of every sac for 1 uterine horn was pooled (2 samples per mouse, right and left horns), and IL-6 levels were quantified by ELISA (BD).