Stimulated PBMCs were incubated with Me290 tumor cells at a tumor cell:PBMCs ratio of 1:10 for 2 h at 37 °C. Brefeldin A (Biolegend, San Diego, CA, USA) was added in the well at 5 ng/mL and incubated for an additional 3 h at 37 °C. The cells were then washed and stained with the following antibodies: anti-CD3 PE/Dazzle 594, anti-CD8 AF700, anti-CD56 BV421, anti-CD16 PE, anti-CD4 PerCP/Cy5.5 (Biologend, San Diego, CA, USA) for 20 min at 4 °C. Cells were then washed, fixed and permeabilized using the Cyto-Fast™ Fix/Perm Buffer Set (Biolegend, San Diego, CA, USA) according to manufacturer instructions and subsequently intracellularly stained with the following antibodies: anti-IFNγ AF647, anti-TNFα FITC and anti-IL2 PE/Cy7 (Biolegend, San Diego, CA, USA) for 20 min at 4 °C. Samples were then analyzed by flow cytometry with a Fortessa instrument.
Anti il2 pe cy7
Manufactured by BioLegend
Sourced in United States
Anti-IL2 PE/Cy7 is a fluorochrome-conjugated antibody that binds to the interleukin-2 (IL-2) cytokine. It can be used to detect and quantify IL-2 expression in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α fitc, by BioLegend (2 mentions)
Cyto fast fix perm buffer set, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Anti cd19 pe, by BioLegend (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd127 pe cy7, by BioLegend (1 mentions)
Anti cd3 percp, by BioLegend (1 mentions)
Anti cd8 pe, by BioLegend (1 mentions)
Anti cd161 fitc, by BioLegend (1 mentions)
Anti ctla4 pe, by BioLegend (1 mentions)
Anti cd15 pe, by BioLegend (1 mentions)
Anti il 8 apc, by BioLegend (1 mentions)
Anti cd163 pe, by BioLegend (1 mentions)
Anti il 17 fitc, by BioLegend (1 mentions)
Anti il 1β fitc, by BioLegend (1 mentions)
Anti tim3 apc, by BioLegend (1 mentions)
Anti ifn γ apc cy7, by BioLegend (1 mentions)
Anti pd 1 fitc, by BioLegend (1 mentions)
Anti il 6 apc, by BioLegend (1 mentions)
Anti ido pe, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti il2 pe cy7
1
Cytokine Production in Stimulated PBMCs
2
Comprehensive Flow Cytometry Immunophenotyping
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For flow cytometry analysis, tumor and blood samples were stained with a cocktail of monoclonal mouse anti-human conjugated antibodies (mAbs): anti-CD45-PercP (clone HI30), anti-CD3-PercP (HIT3a), anti-CD3-APC (UCHT1), anti-CD19-PE (HIB19), anti-CD15-PE (HI98), anti-CD161-FITC (HP-3G10), anti-CD4-FITC (OKT4), anti-CD8-PE (HIT8a), anti-HLA-DR-APC (L243), anti-CD127-PE-Cy7 (A019D5), anti-CD1c-APC-Cy7 (L161), anti-CD163-PE (GHI/61), anti-CD206-APC-Cy7 (15–2), anti-PD-1-FITC (EH12.2H7), anti-PD-L1-APC (29E2A3), anti-CTLA4-PE (L3D10), anti-CD69-APC-Cy7 (FN50), anti-Tim3-APC (F38-2E2), anti-IL-8-APC (E8N1), anti-IFN-γ-PE (4S.B3), anti-IFN-γ-APC-Cy7 (4S.B3), anti-Granzyme B-FITC (QA16A02), anti-IL-1β-FITC (JK1B-1), anti-IL-2-PE-Cy7 (MQ1-17H12), anti-IL-6-APC (MQ2-13A5), anti-IL-17-FITC (BL168), anti-IL-23/IL-12-PE (C11.5), anti-TGF-β-APC (TW4-6H10), all from Biolegend; anti-IDO-PE (eyedio) and anti-IL-10-FITC (BT-10), both from eBioscience; anti-CD25-PE (MEM-181) and anti-CD11b-FITC (LT11) from ImmunoTools.
The antibodies used for immunofluorescence were: mouse monoclonal anti-human CD8 (32-M4) from Santa Cruz Biotechnology and rabbit polyclonal anti-human HLA-DRA from Sigma Aldrich.
The antibodies used for immunofluorescence were: mouse monoclonal anti-human CD8 (32-M4) from Santa Cruz Biotechnology and rabbit polyclonal anti-human HLA-DRA from Sigma Aldrich.
3
Comprehensive T Cell Cytokine Profiling
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Cryopreserved PBMCs from each participant at all time points were investigated simultaneously. After overnight rest of thawed PBMC, the cells were cultured in the presence of anti-CD107a-PE in complete RPMI containing 2 μg/mL of recombinant HBsAg (MyBioSource, USA) and 5 μg/mL of PHA (Sigma-Aldrich, USA) as the positive stimulation control or complete media alone as the unstimulated control at 37°C in a 5% CO2 incubator for 16–18 hr.
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
4
Multiparameter Flow Cytometry Analysis
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Cells were stimulated (3 h) with phorbol-12-myristate 13-acetate (PMA; 50 ng ml−1, Sigma-Aldrich, P1585), ionomycin (1 μg ml−1, Sigma-Aldrich, I3909) and Golgi Stop (1:100, BD Biosciences, 554724). The cells were then fixed and permeabilized (1:100, BD Biosciences, 554714) and stained with anti-IFNγ–APC (1:100, BioLegend, 505810), anti-IFNγ–FITC (1:100, BioLegend, 505806), anti-TNF–BV510 (1:100, BioLegend, 506339), anti-TNF–BV5711 (1:100, BioLegend, 506349), anti-TNF–PE (1:100, BioLegend, 506306), anti-IL2–Pecy7 (1:100, BioLegend, 503832), anti-IL-2–Pacific Blue (1:100, BioLegend, 503820), anti-IL-2–BV510 (1:100, BioLegend, 503833), and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).
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