Splenocytes were isolated and prepared as a single-cell suspension. 3×106 WT and 2B4KO OT-I T cells, respectively, were resuspended in 1.5 mL of complete media (glucose-free RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 0.5 mM 2-mercaptoethanol) in a 24-well flat-bottomed plate at 37°C in a 5% CO2, humidified atmosphere. A D-glucose solution of 200g/L (Gibco by Life Technologies) was titered into the glucose-free complete media and diluted on a half-log scale. Cells were then stimulated with SIINFEKL N4 peptide at 1 nM for 5 days. After 5 days in culture cells were harvested and stained with anti-CD8 (BD Horizon), anti-Thy1.1 (BD Biosciences or BioLegend), anti-CD244 (eBioSciences), anti-Va2 (BD), anti-CD127 (BioLegend), anti-CD62L (BD), anti-KLRG-1, anti-Vb5 (BD), anti-CD44 (BD), 7AAD (BD) and AnnexinV (BioLegend), anti-IL-2 (BD), anti-IFN-g (BD) as described by the manufacturer. As described above, samples were analyzed on an LSRII flow cytometer (BD Biosciences) and data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.).
Anti il 2
Manufactured by BD
Sourced in United States
Anti-IL-2 is a laboratory reagent that binds to and inhibits the activity of the cytokine interleukin-2 (IL-2). It is commonly used in research applications to study the role of IL-2 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti ifn γ, by BD (16 mentions)
Anti cd4, by BD (6 mentions)
Anti tnf α, by BD (4 mentions)
Golgistop, by BD (4 mentions)
Anti il 4, by BD (4 mentions)
Anti ifn γ, by Thermo Fisher Scientific (4 mentions)
Il 12, by Thermo Fisher Scientific (3 mentions)
Anti il 4, by BioLegend (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Anti cd8, by BD (3 mentions)
Anti cd3, by BD (3 mentions)
Anti tnf α, by BioLegend (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Anti ifn γ, by BioLegend (3 mentions)
Golgiplug, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Il 23, by Thermo Fisher Scientific (3 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (3 mentions)
30 protocols using anti il 2
1
Glucose Titration in T Cell Activation
2
Multicolor Flow Cytometric Analyses
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Flow cytometric analyses were performed using the following antibodies: anti-CD4, anti-CD11b, anti-CD33, anti-CD83, anti-CD86, anti-CD206 (BD Biosciences), anti-CD14, anti-CD58, anti-CD80, anti-HLA-DR (ImmunoTools), anti-CD16 (Thermo Scientific), anti-CD25, anti-GARP (eBioscience) and anti-Rab-32 (Abnova).
For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (BD Biosciences). Cytokine expression was analyzed in T cells re-stimulated with 50 ng/ml PMA plus 1 μg/ml Ionomycin for 5 h in the presence of Monensin (1.3 μM) 7 days after in vitro primary stimulation (day 0). Cells were then permeabilized as above and stained with anti-IL-2, anti-IFN-γ or anti-granzyme B mAb (BD Biosciences). Flow cytometry was performed on an LSRII FACS and FACSCalibur (BD Biosciences), using FlowJo software (Tree star).
For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (BD Biosciences). Cytokine expression was analyzed in T cells re-stimulated with 50 ng/ml PMA plus 1 μg/ml Ionomycin for 5 h in the presence of Monensin (1.3 μM) 7 days after in vitro primary stimulation (day 0). Cells were then permeabilized as above and stained with anti-IL-2, anti-IFN-γ or anti-granzyme B mAb (BD Biosciences). Flow cytometry was performed on an LSRII FACS and FACSCalibur (BD Biosciences), using FlowJo software (Tree star).
3
Multiparametric Flow Cytometry Analysis
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FITC-labelled anti-IA/IE, PE-labelled anti-Vβ11, anti-TNFα, anti-CD45.2, anti-CD40, APC-conjugated anti-CD80, anti-IFNγ, anti-CD69, PeCy7-labelled anti-CD11c, PercypCy5.5-labeled anti-CD44, anti-IL-2 and APC-Cy7-conjugated anti-CD62L were obtained from BD Pharmingen (San Diego, CA, USA). PE- labelled anti-CD86, PE-efluor 610-labelled anti-CD25 and Pacific blue conjugated-Annexin V were from Biolegend (San Diego, CA, USA). Alexa 405-conjugated anti-CD4 was obtained from CALTAG lab (Buckingham, UK). APC-conjugated anti-IA/IE and PE-labelled anti-CCR7 were obtained from eBioscience (San Diego, CA, USA). All stainings were performed in presence of mouse Fc Block (BD Biosciences). Dead cells were stained using the Live/Death detection kit with a near-infrared dye (Invitrogen). The samples were analysed using CyAn ADP Analyser (Beckman Coulter, Brea, CA, USA) and the data were analysed using FlowJo version 9.2 (Tree Star inc, Ashland, Oregon, USA).
4
Cytokine-Induced T Cell Activation Assay
5
Comprehensive T-cell Characterization Workflow
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CD8+ T cells (106 mL−1) isolated (Dynabeads, Invitrogen) from cryopreserved PBMC were co-incubated with autologous irradiated CD8+- and CD4+-depleted PBMCs and peptides (1 µg mL−1, single peptide, or pools of ≤ 50 peptides) in RPMI supplemented with 8% human serum and IL-2 (20 IU mL−1 for 48 h and then 100 IU mL−1). IFNγ Enzyme-Linked ImmunoSpot (ELISpot), peptide-MHC multimer complexes staining and ICS assays were performed at day 12. T-cell reactivity for every neo-epitopes was validated by ≥ 2 independent experiments.
ELISpot assays were performed using pre-coated 96-well ELISpot plates (Mabtech) and counted with Bioreader-6000-E (BioSys)33 (link). We considered as positive conditions those with an average number of spots higher than the counts of the negative control (No Ag) plus 3 times the standard deviation of the negative. For ICS, T cells were plated with CD4+ blasts in a 1:1 ratio and brefeldinA (BD biosciences, USA). After 16–18 h, cells were harvested and stained with anti-CD3, anti-CD8, anti-CD4, anti-IL-2, anti-TNFα, anti-IFNγ (BD biosciences) and with viability dye (Life technologies), acquired on a four-lasers Fortessa (BD biosciences), and analyzed with FlowJo X (TreeStar) and SPICE 4.2.334 (link). The number of lymphocyte-gated events ranged between 105 and 106.
ELISpot assays were performed using pre-coated 96-well ELISpot plates (Mabtech) and counted with Bioreader-6000-E (BioSys)33 (link). We considered as positive conditions those with an average number of spots higher than the counts of the negative control (No Ag) plus 3 times the standard deviation of the negative. For ICS, T cells were plated with CD4+ blasts in a 1:1 ratio and brefeldinA (BD biosciences, USA). After 16–18 h, cells were harvested and stained with anti-CD3, anti-CD8, anti-CD4, anti-IL-2, anti-TNFα, anti-IFNγ (BD biosciences) and with viability dye (Life technologies), acquired on a four-lasers Fortessa (BD biosciences), and analyzed with FlowJo X (TreeStar) and SPICE 4.2.334 (link). The number of lymphocyte-gated events ranged between 105 and 106.
6
SARS-CoV-2-S RBD Peptide Stimulation Assay
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Mouse splenocytes were added to the plated (1 × 106/well). A peptide pool consisting of 20-mers (overlapping by 10 amino acids) spanning the SARS-CoV-2-S RBD were synthesized. Mouse splenocytes were stimulated with the peptide pool for 2 hours. The cells were then incubated with GolgiStop (BD Biosciences, USA) for an additional 10 hours at 37°C. Then, the cells were harvested and stained with anti-CD3 (BioLegend), anti-CD8α (BioLegend) and anti-CD4 (BD Biosciences) surface markers. The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences, USA). Half of the cells were stained with anti-IFN-γ (BD Biosciences), anti-IL-2 (BD Biosciences) and anti-TNF-α (BioLegend). Another half of the cells were stained with anti-IL-4 (Biolegend). All labeled lymphocytes were gated on a FACSAriaIII flow cytometer (BD Biosciences, USA).
7
Multi-Parametric Flow Cytometry Analysis
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Labelled anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD44 (IM7.8.1, 1:100), anti-CD62L (MEL14, 1:100), anti-FOXP3 (FJK-16s, 1:100), anti-GATA3 (L50-823, 1:100), anti-IL-2 (JES6-5H4, 1:100), anti-IL-4 (BVD4-1D11, 1:100), anti-IL-17A (TC11-18H10, 1:100), anti-IFN-γ (XMG1.2, 1:100) and specific isotype-matched control antibodies (1:100) were from BD Biosciences, eBioscience or Miltenyl Biotec. Anti-USP21 (G-17, 1:1,000), Anti-Ubiquitin (P4D1, 1:1,000), anti-GATA3 (HG3-31, 1:1,000) was from Santa Cruz Biotechnology. anti-FOXP3 (eBio7979, 1:1,000) was from eBioscience.
8
Antibody-Coated Nanovial Preparation
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Streptavidin-coated nanovials were reconstituted at a five time dilution in Washing Buffer containing 140 nM (20 μg/mL) of each biotinylated antibody or cocktail of antibodies: anti-CD45 (Biolegend, 368534) and anti-IFN-γ (R&D Systems, BAF285), anti-TNF-α (R&D Systems, BAF210), anti-IL-2 (BD Sciences, 555040). Nanovials were incubated with antibodies for 30 minutes at room temperature on a rotator and washed three times as described above. Nanovials were resuspended at a five times dilution in Washing Buffer or culture medium prior to each experiment.
9
Phenotyping Immune Cells via Flow Cytometry
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Isolated monocytes (105) and PBMCs were incubated with 100 ng/ml LPS (Sigma Aldrich), 5 ng/ml IL-2 (Cell Signaling Technologies), 50 ng/ml phorbol-12-myristate-13-acetate (PMA, Merck), and 1 μg/ml ionomycin (Sigma Aldrich) for 37℃, 5 h, respectively; and GolgiStop (BD Biosciences) was added for the final 3 h. Monocytes were stained with FITC-CD14 and PerCP-CD16 (Biolegend), and PBMCs were stained with FITC-CD45RO, PE-CD4, PerCP-CD3, and APC-CCR7 for 30 min. Cells were fixed and permeabilized using the protocol from the Cytofix/Cytoperm kit (BD Biosciences). Then, the monocytes were stained with anti-TNF-α (Biolegend) and anti-IL-6 (Biolegend), and the PBMCs were stained with anti-IFN-γ (BD Biosciences) and anti-IL-2 (BD Biosciences) for 30 min.
Expression of cell-surface markers was assessed using flow cytometry (FACSCalibur, BD, USA) after gating of live cells, and analyzed according to scatter characteristics using FlowJo software.
Expression of cell-surface markers was assessed using flow cytometry (FACSCalibur, BD, USA) after gating of live cells, and analyzed according to scatter characteristics using FlowJo software.
10
Evaluating CD8+ T Cell Interactions
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For 5 h, GP33-specific CD8+ Teff and Tmem were cultured separately or together in RPMI-1640 with 10 ng/mL IL-2 and 0.1 μg/mL GP33 peptide (gift from Marulasiddappa Suresh, University of Wisconsin). Conditioned media was collected by centrifuging wells and collecting the supernatant. For 12 h, GP33-specific CD8+ Teff and Tmem were then co-cultured with B16GP33 melanoma cells with or without conditioned media and anti-IL-2 (BD Biosciences, San Jose, CA) for MTT assays as described above.
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