Blocking studies were performed with cell-free supernatants obtained from freshly isolated NK cells activated overnight in serum-free AIM-V® medium and IL-2 (1.000 U/ml) supplemented with FMKp (10 μg/ml) or poly(I:C)HMW (50 μg/ml). The receptor-blocking was performed by pre-incubating iDC with blocking antibodies for 20 min before their addition into flat-bottom 96-well plates containing the cell-free NK cell supernatant supplemented with IL-4 (500 U/ml) and GM-CSF (500 U/ml). The following receptor blocking antibodies were used: IFNGR1 (20 μg/ml), TNFR1 (20 μg/ml), TNFR2 (20 μg/ml), or isotype control (all purchased from R&D systems). The blocking of the cytokines (IFN-γ and TNF-α) in the NK cell-derived supernatants was performed by pre-incubating the supernatants with anti-TNF-α (20 μg/ml; BD) or anti-IFN-γ (10 μg/ml; BD) before adding the iDC. As reference value, iDC were incubated with NK cell-derived supernatant in the absence of blocking agents. As a negative control, iDC were incubated with medium supplemented with FMKp or poly(I:C) and IL-2 (control ‘supernatant’). After 48 h of maturation, the supernatant was harvested to determine the DC cytokine and chemokine profiles.
Tnfr2
Manufactured by R&D Systems
Sourced in United States
TNFR2 is a recombinant protein that functions as the extracellular domain of the human tumor necrosis factor receptor 2 (TNFR2). It is a member of the tumor necrosis factor receptor superfamily and is involved in the regulation of various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Tnfr1, by R&D Systems (6 mentions)
Isotype control, by R&D Systems (1 mentions)
Anti ifn γ, by BD (1 mentions)
Ifngr1, by R&D Systems (1 mentions)
Anti tnf α, by BD (1 mentions)
Caspace fitc vad fmk in situ marker, by Promega (1 mentions)
Tsa plus kit, by PerkinElmer (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Bz 9000, by Keyence (1 mentions)
Tunel kit, by Roche (1 mentions)
Bz 8100, by Keyence (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
E cadherin, by R&D Systems (1 mentions)
Gal 9, by R&D Systems (1 mentions)
Tim 3, by R&D Systems (1 mentions)
Tnf α, by BioLegend (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
8 protocols using tnfr2
1
NK Cell Supernatant-Mediated Dendritic Cell Maturation
2
Immunohistochemical Analysis of Intestinal Tissues
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Cryo-frozen intestinal cross-sections were fixed with 4% paraformaldehyde (PFA) and stained using the TSA plus kit (Perkin Elmer, Baesweiler, Germany) according to the manufacturer’s instructions. Alternatively, slides were fixed with methanol at −20°C for 10 min, blocked with 10% fetal calf serum (FCS)/1% bovine serum albumin (BSA) for 1 hour and incubated overnight at 4°C with the primary antibody. Paraffin-embedded tissues were deparaffinised and antigen unmasking was performed using citrate buffer. After blocking, slides were incubated with TNFR2 (R&D, Minneapolis, Minnesota, USA), pSTAT3 (Cell Signalling, Leiden, Netherlands), CD3 (BD Biosciences, Heidelberg, Germany), Ki-67 (eBioscience, Frankfurt, Germany) or CD14 (Abcam, Cambridge, UK) antibodies. From each sample, 3–6 high power fields (HPF) per patient were analysed using ×10 objective magnification. For staining of active Caspase, the CaspACE FITC-VAD-FMK In Situ Marker (Promega, Mannheim, Germany) was used. TUNEL staining of PBMCs was performed with the TUNEL kit (Roche Diagnostics, Mannheim, Germany). Analysis of images was done with a fluorescence microscope (BZ-8100 or BZ-9000; Keyence, Neu-Isenburg, Germany) or a confocal microscope (LSM; Leica Microsystems, Wetzlar, Germany).
3
Cytokine Profiling in MDM Infection
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Culture supernatants of 1 × 106 MDMs/mL (per condition) were collected after 24 h of infection and stored at −70 °C until the analysis. The concentrations of TNF, TNFR1, TNFR2, IL-10, IP-10, CCL2, IL-8, and CCL19 were measured using a sandwich-type immunoassay (ELISA) according to the manufacturer’s instructions. Briefly, ELISA microplates with 96 wells were coated overnight at 4 °C with one of the capture antibodies specific to TNF, IL-10, IFN-γ, CCL2, IL-8 (BioLegend, San Diego, CA, USA), TNFR1, or TNFR2 (R&D Systems, Minneapolis, MN, USA); for CCL19, a kit with pre-coated 96-well microplates was used (Thermo Fisher Scientific, Waltham, MA, USA). Plates were blocked with 1% FBS in PBS for 1 h at room temperature (RT), after which the supernatants were added and incubated for 2 h at RT. Each protein was recognized using a specific biotinylated antibody. Quantification was performed using avidin–HRP conjugate for each ELISA kit, and the tetramethylbenzidine colorimetric substrate was used to develop the blue color. The optical density (450 nm) was measured using a microplate reader (Imark, Bio-Rad, Hercules, CA, USA).
4
Osteogenic Differentiation of Murine BMSC
5
Monocyte Phenotyping by Flow Cytometry
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After separating monocytes, cells were stained with fluorescence-bound antibodies for CD14, TNFR1, TNFR2 and CD54 (R&D Systems, Minneapolis, MN, USA) and analyzed on a FACSCalibur system.
6
Quantifying Immune Biomarkers in Plasma
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Plasma samples were stored at −70 °C until analysis. Soluble levels of TIM-3, Gal-9, E-cadherin, TNFR1, TNFR2 (provided by R&D Systems, Minneapolis, USA), TNF-α, IFN-γ, IL-6 (provided by BioLegend, San Diego, CA, USA), ADAM10, and ADAM17 (provided by Cloud-Clone Corp., Houston, TX, USA) were quantified by ELISA following the manufacturer’s protocols. All proteins were quantified by comparison with the corresponding standard curve.
7
Measurement of Inflammatory Markers
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Soluble markers of inflammation were measured with ELISA-based assays. Plasma was analyzed for levels of IL-6, sCD14, IP10, sCD163, CRP, TNFR1 and TNFR2 (all from R&D Systems, Minneapolis, MN), D-dimer (Diagnostica Stago, Parsippany, NJ), and IL10 and TGFβ (both from MSD, Rockville, MD) per the manufacturers’ protocols. Since these markers were measured in two batches, we examined the batch effect of the overlapping tests and did not find batch effect (SI Appendix, Fig. S7 ).
8
Multicolor Immunophenotyping of Cell Subsets
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Cells were stained for 20 min at 4°C with fluorochrome-conjugated mAb against CD14, TIM3, GAL9, TNF, and interleukin-1 receptor (IL-1R) (BioLegend, San Diego, CA, USA). Both TNFR1 and TNFR2 (R&D Systems, Minneapolis, MN, USA). After incubation, cells were washed and re-suspended in staining buffer (BD Biosciences, San Jose, CA, USA) prior to FACS analysis. Data were collected using a FACS Aria II flow cytometer (Becton Dickinson, San Jose, CA, USA) and FACS Diva software (V.8.01). Cells were then analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA). Typically, 20,000 events were acquired.
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