410 mt 010 cf

MKTAL is a thick ascending limb cell line that was isolated and characterized by Bourgeois et al. [21 (link)]. Cells are cultured in DFS, which consists of 1:1 mixture of DME (Gibco #10567–014) and Ham’s F12 (Gibco #31765–035) medium containing 5% FBS (Lonza BioWhittaker #14–502F). Experiments were performed when cells were 70–80% confluent. All experiments were performed in triplicates, and representative experiments are shown for each experimental condition. TNFα (R&D Systems #410-MT-010/CF) was added at 1, 10, and 20 ng/ml as indicated. Control wells without TNFα were used for each time point. After 2, 6, or 24 h of incubation, flasks from each group were aspirated, trypsinized, and harvested as above.

Cells were treated with 0.1 or 1 ng/mL mouse (400-ML-005/CF, R&D Systems) or human (200-LA-002/CF, R&D Systems) IL-1α, 1 ng/mL mouse IL-1β (401-ML-005/CF, R&D Systems), 10 ng/mL mouse TNF-α (410-MT-010/CF, R&D Systems), 20 ng/mL mouse (7666-MB-005/CF, R&D systems) or human TGF-β1 (T7039-2UG, Sigma), 500 nM JAK inhibitor AZD1480 (S2162, Selleck Chem), 30 μM IKK-β inhibitor ML102B, 1 μM A83-01 (2939, Tocris Bioscience), 3 μg/mL IL-1α neutralizing antibody (MAB4001, R&D Systems) or an IgG control (400902, Biolegend), 5 μg/mL TNF-α neutralizing antibody (11969S, Cell signaling) or an IgG control (sc-2027, Santa Cruz), 3.4 μg/mL LIF neutralizing antibody (AF449, R&D Systems) or an IgG control (AB-108-C, R&D), 3.8 μg/mL G-CSF (MAB414, R&D Systems) or IL-6 (MAB406, R&D Systems) neutralizing antibodies or an IgG control (MAB005, R&D).