Recombinant human tnf α

The Luciferase reporter gene assay was performed as previously described (Hale et al., 2010 (link)) with minor modifications. Briefly, 293T cells were seeded in 6-well plates (Greiner; 2 × 10⁶ 293T cells/well) and co-transfected with 1 μg of either pMP-NS-Gi, pMP-NS-PR8, pMP-NS-Vict or empty pHW2000 plasmids mixed with 40 ng pRL-SV40 (Renilla luciferase expression plasmid), and 200 ng p125-Luc (Firefly luciferase plasmid) (Yoneyama et al., 1998 (link)), which contains the luciferase reporter gene under the control of the IFN-β promoter. Transfection was performed using Trans-IT2020 as previously described (Mostafa et al., 2013 (link)) for 8 h. Except for the mock, transfected cells were stimulated either by infection with virulent Newcastle disease virus (NDV, strain Herts 33/56) at MOI = 3 or treated with DMEM containing 50 ng/ml of recombinant human TNF-α (Invitrogen). After 24 h of stimulation, the cells were harvested, washed one time with 1x PBS, and lysed with 200 μl of “1x passive lysis buffer” (Promega). The amount of Firefly/Renilla luciferase was quantified using the Dual-Luciferase Reporter Assay System (Promega) and measured using a Spark 10M multimode microplate reader (TECAN). Relative luminometer units (RLU), normalized to Renilla luciferase, refer to fold induction of IFN-β promoter activity.

Recombinant human TNF-α (purchased from Invitrogen) was added to complete RPMI-1640 media at either 5 or 2.5 ng/mL. GelMA hydrogels were made as described above in the GelMA hydrogel preparation section. Five hundred microliter of media with or without the addition of TNF-α was incubated with the gels for 4 h in a humidified incubator (37°C, 5% CO₂). Supernatant was collected and stored at −80°C for cytokine analysis.

Wells of Angiogenesis ibiTreat chamber slides (Thistle Scientific, UK) were coated with 10 μl growth factor reduced Matrigel (BD Biosciences, Oxford, UK), and incubated at 37 °C in an atmosphere of 5% CO₂ for 30 min. 2 × 10⁴ SGHEC-7 cells/well were added (50 μl per well), and allowed to form tube-like structures over 6 h. The SGHEC-7 culture medium was then removed and replaced by 50 μl of pooled normal-RI or high-RI dNK cell conditioned medium (n = 18–19). The median gestational age was 11.3 weeks (range 9.3–13.9) for normal-RI and 10.8 weeks (range 9.1–13.7) for high-RI cells used to generate pools of conditioned medium, p = 0.4, t-test. To investigate whether dNK-derived factors were inducing EC apoptosis, 50 μM zVAD-fmk, a broad spectrum caspase inhibitor (Calbiochem, UK), was added with normal-RI dNK-conditioned medium. To investigate the effect of TNF-α 20–50 ng/ml recombinant human TNF-α (Peprotech, UK) was added to the preformed EC structures. Images were captured at 0, 2, 5, 10 and 18 h, using an Olympus 1 × 70 inverted microscope (Olympus, Tokyo, Japan) with a Hamamatsu C4742-95 digital camera (Hamamatsu Protonics, UK). At each time point, the length of the EC tube-like structures in duplicate positions of the well were analysed using Image Pro plus software (Media Cybernetics, MD).

Experiments were performed as previously described [23 (link)]. Briefly, experiments were initiated when HT-29 cells were at confluence (∼1.83×10⁶ cells/well). LAB were added at MOI 40:1 in 50 μL DMEM in a total volume of 500 μL. Cells were stimulated simultaneously with recombinant human TNF-α (5 ng/mL; Peprotech, NJ, USA) for 6 h at 37°C in 10% CO₂. Stimulation of the HT-29 cell line with TNF-α alone was used as the reference condition. All samples were analyzed in triplicate in three independent assays (a total of nine data points). After coincubation, cell supernatants were collected and mixed with anti-protease cocktail, as indicated by the manufacturer (Complete, EDTA-free tablets, Roche) and frozen at −80°C until further analysis of IL-8 concentrations by ELISA (Biolegend, San Diego, CA, USA).

Antibodies and dilution conditions are presented in Table 1. Recombinant human IL1β (211‐11B; PeproTech, Rocky Hill, NJ, USA) and recombinant human TNFα (300‐01A; PeproTech) were prepared according to the manufacturer's instructions.

Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), enzymes from New England Biolabs (Ipswich, MA, USA), and culture medium from Gibco Invitrogen (Carlsbad, CA, USA). Lipofectamine 2000 and Trizol reagents were purchased from Invitrogen (Carlsbad, CA, USA). The nucleoprotein and cytoplasm-protein extraction kit was obtained from FD Bioscience (Hangzhou, Zhejiang, China). Recombinant human TNF-α was obtained from Peprotech (Rocky Hill, NJ, USA). The peptide was first diluted in water as stock and further diluted in medium containing bovine serum albumin at a final concentration of 20 μg/ml.