The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described23 (link). Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 105 CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).
Anti cd3 pe cf594
Manufactured by BD
Sourced in United States, France
Anti-CD3 PE-CF594 is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen expressed on the surface of human T cells. It can be used in flow cytometry applications for the identification and analysis of T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd107a af488 antibody, by BioLegend (4 mentions)
Live dead fixable aqua dead cell staining kit, by Thermo Fisher Scientific (4 mentions)
Anti ifn γ bv 421, by BioLegend (4 mentions)
Brefeldin a, by BD (4 mentions)
Monensin, by BD (4 mentions)
Anti cd56 apc, by BD (4 mentions)
Anti ly6g apc cy7, by BioLegend (4 mentions)
Anti cd56 pe cy7, by BioLegend (4 mentions)
Live dead fixable violet dead staining kit, by Thermo Fisher Scientific (3 mentions)
Live dead reagent, by Thermo Fisher Scientific (3 mentions)
Fortessa, by BD (2 mentions)
Anti cd4 fitc, by BioLegend (2 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Diva software, by BD (2 mentions)
Anti cd206 apc, by BioLegend (2 mentions)
Facs fortessa flow cytometer, by BD (2 mentions)
Anti cd19 pe cf594, by BD (2 mentions)
Anti cd49b pe cf594, by BD (2 mentions)
Anti cd45 bv605, by BD (2 mentions)
Anti cd11b percp cy5, by BioLegend (2 mentions)
20 protocols using anti cd3 pe cf594
1
Evaluating NK Cell Activity Assay
2
Production and Characterization of Monoclonal Antibodies
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The following primary mAbs were produced in our laboratories: M5A10 (IgG1, anti-PVR), L14 (IgG2a, anti-nectin-2), BAM195 (IgG1, anti-MICA), A6136 (IgM, anti-HLA class-I), NE97 (IgG2b, anti-B7-H3) (25 (link)), BAB281 (IgG1, anti-NKp46) (8 (link)). Anti-PD-L1.3.1 (IgG1, anti-PD-L1), and anti-PD-L2 (IgG1) were produced in D. Olive lab. D1.12 (IgG2a anti-HLA-II) mAb was produced by R. Accolla (University of Insubria, Varese, Italy). Anti-ULBP1-4 were purchased from R&D System (Minneapolis, US) and Santa Cruz Biotecnology (Dallas, US); anti-GD2 (IgG2a) was purchased from BD Bioscience PharMingen, San Diego, CA); anti-CXCR4 (IgG2b) was purchased from &D Systems (Minneapolis, MN).
The following fluorochrome-conjugated anti-KIR in combination with anti-CD3-PECF594 (IgG1; BD Bioscience) and anti-CD56-PC7 (IgG1; Beckman Coulter, Marseille, France) mAbs were used: GL-183 (IgG1, anti-KIR2DL2/L3/S2; Beckman Coulter), EB6B (IgG1, anti-KIR2DL1/S1 and KIR2DL3*005; Beckman Coulter), Z27 (IgG1, anti-KIR3DL1/S1; Beckman Coulter), DX9 (IgG1, anti KIR3DL1; Miltenyi Biotec, Bergisch Gladbach, Germany), FES172 (IgG2a, KIR2DS4; Beckman Coulter), 143211 (IgG1, anti-KIR2DL1 and KIR2DS5; R&D). ECM41 mAb (IgM, anti-KIR2DL3, with the exception of *005; Our laboratory) followed by FITC-conjugated goat anti-mouse IgM (Southern Biotech, Birmingham, AL) was also used.
The following fluorochrome-conjugated anti-KIR in combination with anti-CD3-PECF594 (IgG1; BD Bioscience) and anti-CD56-PC7 (IgG1; Beckman Coulter, Marseille, France) mAbs were used: GL-183 (IgG1, anti-KIR2DL2/L3/S2; Beckman Coulter), EB6B (IgG1, anti-KIR2DL1/S1 and KIR2DL3*005; Beckman Coulter), Z27 (IgG1, anti-KIR3DL1/S1; Beckman Coulter), DX9 (IgG1, anti KIR3DL1; Miltenyi Biotec, Bergisch Gladbach, Germany), FES172 (IgG2a, KIR2DS4; Beckman Coulter), 143211 (IgG1, anti-KIR2DL1 and KIR2DS5; R&D). ECM41 mAb (IgM, anti-KIR2DL3, with the exception of *005; Our laboratory) followed by FITC-conjugated goat anti-mouse IgM (Southern Biotech, Birmingham, AL) was also used.
3
ADCC Assay for NK Cell Function
4
Identification of Tumor-Infiltrating Immune Cells
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Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
5
Liver Immune Cell Subset Profiling
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Characterization of immune cell subsets in the liver was performed essentially as described previously21 (link). The individual samples were analyzed with a LSRII/Fortessa flow cytometer (BD Biosciences) and the FlowJo software Vx (Treestar). All indicated antibodies and reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using cytometer setup and tracking beads (BD). Single cell suspensions were created using the Miltenyi Liver Dissociation Kit (No. 130-105-807) and the GentleMACS isolator (Miltenyi) using standard protocols. The following antibodies were used: anti-CD3-PE-CF594, anti-CD4-V500, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326 (EPCAM)-BV711, anti-Ly6C-PerCP-Cy5.5 (all from BD), anti-CD8-eFluor650, anti-CD11b-eFluor605NC (eBioscience), anti-CD45-VioBlue, anti-CD49b-PE, anti-MHC-II-APC (Miltenyi), anti-F4/80-PE-Cy7, anti-Ly6G-APC-Cy7 (Biolegend). A gating strategy is provided in the supplementary material and methods (Fig. S1 ).
6
Comprehensive Immune Profiling of Tumor and Spleen Cells
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Tumor cells and spleen cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described previously (46 (link)). Briefly, tumor single-cell suspensions were stained with the LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher Scientific, Inc.) and then labelled with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 Alexa Fluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD80 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE (all from BioLegend) for lymphocyte identification. Then, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. In spleen single cell suspension only the lymphocyte identification was performed according to the procedure described above. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
7
T Cell Receptor Characterization via Tetramer Staining
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STCL were generated as above using a single “parental” 15-mer peptide. To examine optimal peptide length and their HLA restriction, 721.221 or C1R cells transfected with a single HLA class I allele were pulsed with short peptides and used as antigen-presenting cells for stimulation of STCL effectors in an ICS assay, which also confirmed that the responding T cells were CD8+ [62 (link)]. For tetramer reactivity, 106 STCL cells were stained with pre-titrated amounts of PE-conjugated tetramer (NIH Tetramer Facility, Emory University Vaccine Center, Atlanta, GA, USA) in FACS tubes at room temperature for 10 min. For both HLA and tetramer procedures, a mix of anti-CD8 FITC, anti-CD4 PE, anti-CD3 PE-CF594, anti-IFN-γ V450 (BD Biosciences, Wokingham, UK), and anti-CD3 ECD (Beckman-Coulter, High Wycombe, UK) monoclonal antibodies (mAbs), and LIVE/DEAD fixable cell stain Aqua were added and the tubes incubated for a further 20 min at room temperature. The cells were washed with FACS buffer, fixed with 1% paraformaldehyde prior to analysis, acquired by a Fortessa flow cytometer (Becton-Dickinson, Franklin lakes, NJ, USA) and analyzed using the FlowJo software (Tree Star).
8
Assessing TriKE-Mediated NK Cell Expansion
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To measure the ability of the TriKE to specifically induce NK cell expansion via the IL-15 moiety, PBMCs from healthy donors were labeled with CellTrace Violet Proliferation Dye (Invitrogen, Carlsbad, CA, USA) according to kit specifications. After staining, cells were cultured with TriKEs at noted concentrations, or equimolar concentrations of controls, and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for seven days. Cells were harvested, stained for viability with Live/Dead reagent (Invitrogen, Carlsbad, CA, USA) and surface stained for anti-CD56 PE/Cy7 (Biolegend, San Diego, CA, USA) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ, USA) to gate on the viable CD56+ CD3- NK cell population or the CD56-CD3+ T cell population. Data analysis was performed using FlowJo software (FlowJo LCC, version 7.6.5, Ashland, OR, USA).
9
NK Cell-Mediated ADCC Assay
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The ADCC assay measuring intracellular NK cell IFNγ and CD107a expression was conducted as previously described with minor modifications.35 (link) Briefly, 96-well plates were coated overnight at 4 °C with 1 μg/ml HA1 (A/California/06/2009(H1N1)) 6×His tagged (eEnzyme, USA) or chimeric cH6/1 in PBS. Plates were washed with PBS and incubated with heat-inactivated human sera for 2 h at 37 °C. After washing, 105 CD16 176v NK-92 cells (mycoplasma-free, human NK cell line expressing high affinity 176V variant CD16 receptor) (kindly provided by Fox Chase Cancer Center, Philadelphia, PA, USA) were added per test well. As a negative control for each sample, NK-92 cells (lacking expression of CD16) were added to an additional well. The cells were incubated for 16 h at 37 °C with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA, 328610), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD). Cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen, Carlsbad, CA, USA), anti-CD3-PE CF594 (BD, Franklin Lakes, NJ, USA, 562280) and anti-CD56-APC (BD, 555518) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend, 502532). Cells were acquired on BD Fortessa (San Jose, CA, USA). Data analysis was done using FlowJo version 10 (treeStar, Ashland, OR, USA).
10
Intracellular Cytokine Staining and Vaccinia Infection
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For intracellular cytokine staining, lymph node T cells or cells after primary ConA activation and IL-2 expansion were stimulated (1 h, 37°C) in medium with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (2 μg/ml). Brefeldin A (GolgiPlug, 20 μl/ml; BD PharMingen) was added and cells incubated (3 h, 37°C). After surface marker staining with appropriate combinations of labeled anti-CD4, -CD8, -Thy1, -CD44, and -CD62L, cells were washed with PBS, fixed and permeabilized with Cytofix/Cytoperm (100 μl, 20 min, 4°C), washed once with Perm/Wash buffer and blocked with 150 μl Perm/Wash buffer + 1% BSA (30 min, room temperature (RT)). Anti-IL-17-PE, -IL-2-PE and -IFNγ-PE antibodies were added (all 1/100, PharMingen); permeabilized cells were incubated (20 min, 4°C) and washed twice in Perm/Wash before FACS analysis.
For the vaccinia infection experiment, splenocytes were stained for surface markers with anti-CD3-PE-CF594, -CD4-APC-Cy7, -CD8-V500 (all from BD), fixed, permeabilized (Cytofix/Cytoperm kit; BD), and stained intracellularly with anti-IFNγ-PE, and -TNFa-PE-Cy7 (from BD). Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).
For the vaccinia infection experiment, splenocytes were stained for surface markers with anti-CD3-PE-CF594, -CD4-APC-Cy7, -CD8-V500 (all from BD), fixed, permeabilized (Cytofix/Cytoperm kit; BD), and stained intracellularly with anti-IFNγ-PE, and -TNFa-PE-Cy7 (from BD). Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).
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