Ab46100

For cytokine measurements, primary mixed glial cells were stimulated, and the supernatant was subjected to ELISA analysis for mouse TNFα (Abcam; #ab208348, Cambridge, MA, USA), IL-6 (Abcam; #ab46100, Cambridge, MA, USA) and IL-10 (Abcam; #ab100697, Cambridge, MA, USA). The absorbance read was performed using a microplate reader at 450 nm, and the results were compared against a standard curve. Furthermore, the DICAM levels of cultured supernatant and plasma were measured using the DICAM ELISA kit (MyBioSource; #MBS907149, San Diego, CA, USA) according to the manufacturer’s instructions.

TCM was obtained from 5×10⁶ total tumor cells that underwent single-cell suspension and were subsequently cultured for 24 hours in serum-free medium. TCM from met-low or met-high B16 melanoma tumors was applied to a proteome profiler mouse XL cytokine array (ARY028; R&D systems) in accordance with the manufacturer’s instruction. The signals corresponding to each factor in the array were quantified by densitometry analysis. The ratio between the levels of the various factors in met-high and met-low TCM was calculated. IL-6 levels in TCM of met-low and met-high melanoma and breast cancers were quantified using a specific ELISA kit (ab46100; Abcam). The ELISA experiments were performed with five to eight mice per group and analyzed as mean±SD

Maternal or fetal blood samples were collected into precooled tubes containing EDTA or heparin centrifuged at 2200 g for 15 min at 4 °C. The supernatants (plasma) were stored at −80 °C until analysis. Amniotic fluid or fetal blood samples were collected from multiple fetuses derived from one dam (a dam was considered as n = 1). A Mouse Inflammatory Cytokines Multi-Analyte ELISArray Kit (MEM-004A; SABiosciences, Germantown, MD, USA) was used to detect 12 types of proinflammatory cytokines in the plasma or amniotic fluid sample. The cytokines in the array were interleukin (IL)-1A, IL-1B, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon-γ, tumor necrosis factor-α (TNF-α), granulocyte colony-stimulating factor (G-CSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF). Only positivity or negativity, according to the optical density at 600 nm, could be detected. For further analysis, the concentration of IL-6 (ab46100; Abcam, Cambridge, UK) was measured according to the manufacturer’s protocol.