Anti tnf α fitc

To assess the frequencies of IFN-γ+ and TNF-α in CD8+ T cells, tumor cells were fixed, permeabilized, and stained by anti-CD8-APC, anti-IFNγ-FITC, or anti-TNF-α-FITC (BD Biosciences, San Jose, California, USA). Then, staining with fluorophore-conjugated secondary antibodies was used for flow cytometry analysis.

After stimulation, 2 mM EDTA (final concentration) was added to each well for 10 min at room temperature (RT). A LIVE/DEAD Fixable Dead Cell Stain (blue fluorescent reactive dye; Invitrogen) was used to identify and exclude non-viable cells from the analysis. Cells were fixed (BD Lyse Solution) and permeabilized (BD Perm 2), according to manufacturer's instructions. Surface and intracellular staining were done simultaneously for 30 min at RT with the following pre-titered antibody panel: anti–CD3 PerCP Cy5.5 (UCHT1; BD), anti–CD8 V450 (RPA-T8; BD), anti–CD4 V500 (RPA-T4; BD), anti–IFNγ PE-Cy7 (B27; BD), anti–IL-2 PE (5344.111; BD), anti–TNFα FITC (6401.1111;BD), and anti–MIP-1β APC (D21-1351; BD). Cells were acquired using an LSRII flow cytometer (BD) within 24 hours and ≥200,000 total events were collected for each sample unless otherwise specified.

Ablated tumor cells or splenocytes (5×10⁵) were prepared for immunostaining for fluorescence-activated cell sorting (FACS) analyses. Anti-mouse calreticulin-phycoerythrin (PE) and matched isotype control antibody were obtained from R&D Systems (Minneapolis, MN, USA). Anti-mouse Fas ligand–fluorescein isothiocyanate (FasL-FITC) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-PE were from BD Biosciences (Franklin Lakes, NJ, USA). For staining of surface markers, cells were washed with FACS staining buffer (PBS containing 0.2% FBS) and resuspended in 100 μL of staining buffer containing the corresponding antibodies, followed by incubation at 4°C for 1 h. After washing twice with FACS staining buffer, cells were fixed in 200 μL of 1% paraformaldehyde.
For intracellular staining of interferon (IFN)-γ and TNF-α, after surface staining with anti-mouse CD3-AF700, CD4-V450, and CD8-PerCP antibodies (BD Biosciences), cells were permeabilized in 100 μL of Fixation/Permeabilization Solution (BD Biosciences) for 20 min at 4°C, washed with Permeabilization/Washing buffer (BD Biosciences), and then underwent intracellular staining with anti-IFN-γ-PE and anti-TNF-α-FITC (BD Biosciences). Cellular fluorescence was examined on a FACSCalibur instrument (BD Biosciences) and analyzed using CellQuest (BD Biosciences).

For the measurement of intracellular cytokine production, a separate aliquot of PBMCs was incubated with R10 (unstimulated), PMA and ionomycin (n = 40) or α-GalCer (n = 20), as described above. After 1 h at 37°C in 5% CO₂, Brefeldin A (BRFA) (10 µg/mL; Sigma-Aldrich, Milan, Italy,) was added. After incubation for 14 h, the cells were washed and surface stained as described above, with the exception of anti-CD161. Cells were washed again and incubated with 1 mL of FACS Lysing Solutions (BD Bioscences, San Jose, California, USA) for 45 minutes at room temperature, in the dark and washed prior to intracellular staining with anti-TNF-α-FITC (BD Bioscences, San Jose, California, USA)/IFN-γ-FITC (Beckman Coulter, Fullerton, California, USA). After 30 minutes of incubation at 4°C in the dark, samples were washed, run on a FACS CANTO 2.6 cytometer and analyzed with FACS Diva 6.1.3 software.

Surface and intracellular staining for flow cytometry analysis was performed as described previously (46 (link), 47 (link)). For surface staining, cells were incubated with relevant fluorochrome-labeled antibodies for 30 min at 4°C in the dark. For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization buffer set (Invitrogen, USA) and stained with allophycocyanin (APC)-anti-IFN-γ, peridinin chlorophyll protein (PerCP)-Cy5.5-anti-IL-2, or fluorescein isothiocyanate (FITC)-anti-TNF-α (BD Biosciences, USA). Freshly isolated cells were used for all assays. Approximately 100,000 PBMCs were acquired for each sample using a BD FACS Canto II flow cytometer. Data analysis was performed using FlowJo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506.

Isolated cells in the lung were stained with indicated antibodies for further analysis. For immunostaining, the cells were washed two times with PBS containing 2% FBS, adjusted to approximately 1 × 10⁵ to 1 × 10⁶ cells in 100 μL of the same buffer, and labeled with PE-anti-F4/80 (BD Bioscience, 565410), PE-Cy7-anti-CD11b (BD Bioscience, 561098), and FITC-anti-TNF-α (BD Bioscience, 554418). Incubations with antibodies were performed for 30 min at 4 °C in the dark, and then cells were centrifuged for 4 min at 900 rpm. After removal of the supernatant, the cell pellet was washed once with PBS containing 2% FBS and then resuspended in fixation and permeabilization buffer (BD Bioscience, Franklin Lakes, NJ, USA) for 30 min at 4 °C. Cells were washed two times with PBS containing 2% FBS and then acquired by FACSVerse (BD Bioscience) and analyzed using software Flow Jo (Tree Star, Inc., Ashland, OR, USA).