Anti tnf α apc

The following Abs were used for short-term culture or surface marker and ICS for flow cytometry (all Abs were from Biolegend): anti-CD3-PerCP-cy5.5, anti-CD8-APC-Cy7, anti-CD4-PE-Cy7, anti-CCR5-PE, anti-CXCR3-APC, anti-CXCR4-PE, anti-CCR7-APC, anti-granzyme A-PE, anti-Perforin-APC, anti-IFN-γ-PE, and anti-TNF-α-APC. The reagents listed below were all commercial products: brefeldin A (GolgiPlug, BD Biosciences), Cytofix/Cytoperm (BD Biosciences), and Perm buffer (BD Biosciences).

These following antibodies (Abs) were used for surface marker staining and ICS combined with flow cytometry (all Abs were from Biolegend): anti-CD3-PErCP-cy5.5 (UCHT1), anti-CD3-FITC (UCHT1), anti-CD3-PE (HIT3a), anti-CD4-APC (OKT4), anti-CD4-APC-cy7 (RPA-T4), anti-CD8-PE (RPA-T8), anti-CD8-APC-cy7 (SK1), anti-CD14-FITC (HCD14), anti-IFN-γ-PE (4S.B3), anti-TNF-α-APC (MAb11), anti-IL-17A-PE-cy7 (BL168), anti-Foxp3-PE (206D), anti-TGF-β-PE-cy7 (TW4-2F8), anti-Perforin-PC (dG9), anti-granzyme A-PE (CB9),.

IFN-γ and TNF-α production by NK cells was evaluated in all samples available for phenotypic analysis after overnight stimulation with or without IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA) added during the last 3 h of culture. Surface staining with anti-CD3-PE, anti-CD56-PE-CF594, and anti-CD16-FITC (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Then, cells were fixed with medium A reagent and permeabilized with medium B reagent (Nordic Mubio, Lifespan Biosciences, Seattle, WA, USA) in accordance with manufacturer’s instructions. Cytokine determinations were performed by intracellular cytokine staining (ICS) with anti-IFN-γ-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA) and anti-TNF-α-APC (BioLegend, San Diego, CA, USA) monoclonal antibodies and analyzed by flow cytometry. The CD107a degranulation assay was performed to assess the cytotoxic potential. Cells were stimulated overnight with IL-12 and IL-18 (as above), then incubated with K562 target cells for the last 4 h in the presence of BFA and anti-CD107a-PE-Cy7 (BD Bioscience, Franklin Lakes, NJ, USA).
Data are expressed as the difference between the percentage of cytokine or CD107a-positive⁺ NK cells in the stimulated and unstimulated samples.

To assess apoptosis, the cells were cultured for 24 h in RIPM 1640 medium containing normal glucose (2 g/L) or low glucose (40 mg/L) levels. The cells were centrifuged and resuspended in 0.5 ml annexin V-binding buffer (KeyGEN Biotech, China). Thereafter, 5 μl annexin V-APC and 7-AAD were added to the samples and incubated at RT for 10 min in the dark. The samples were then analyzed on a FACS Caliber flow cytometer (BD Biosciences, US).
The lymphocytes from 4T-1-injected BALB/c mice were isolated as follows: the tumor tissues from the mice were sectioned and digested with 2 mg/ml collagenase IV and 100 ng/ml DNase I (sigma) at 37 °C for 30 min. The tissues were then added to RPMI 1640 media supplemented with 10% FBS and 0.5 mM EDTA and separated by discontinuous 30–70% Percoll (GE Healthcare). After stimulation with PMA/Ionomycin and BFA (sigma) for 6 h at 37 °C, the cells were harvested for surface staining and intracellular staining (BD Pharmingen), according to the manufacturer’s instructions. The antibodies were anti-CD45-BV510, anti-CD45-APCcy7, anti-CD3-APC, anti-CD3-BV650, anti-CD4-FITC, anti-CD4-PEcy7, anti-PD-1-BV785, anti-PD-1-PE, anti-TIGIT-BV421, anti-TCR α/β-PE anti-IFN-γ-BV785, anti-IFN-γ-APC, anti-TNF-α-BV421 and anti-TNF-α-APC (BioLegend). All the data were collected by BD FACS Celesta flow cytometry and processed by Flow Jo software.