Splenocytes from mice at up to 5 × 106 cells were seeded together with 5 mg/mL mouse IL-2 (Biolegend), 1 μL BD GolgiPlug (Becton Dickinson, Franklin Lakes, NJ) or Brefeldin A (ThermoFisher Scientific) and 10 μg peptide in 96-well tissue culture plates, followed by incubation at 37°C, 5% CO2 for 5 h. For Plasmodium studies, the mouse IL-2 addition was omitted. They were then stained with Zombie Aqua Fixable Viability kit (Biolegend) for 30 min, washed and followed by antibody stainings for 30 min, selected from the following panel: purified CD16/32 (clone 93); CD3 PE/Dazzle 594 (clone 17A2); CD3ε Brilliant Violet 421 (clone 145-2C11); CD4 APC/Cy7 (clone GK1.5); CD8a PerCP/Cy5.5 (clone 53-6.7); CD8b PerCP/Cy5.5 (clone YTS 156.7.7, all from Biolegend). After washing, cells are fixed in 4% formaldehyde, permeabilized with 0.1% saponin (Sigma-Aldrich) or per buffer (BD Bioscience), followed by staining with IFNγ FITC (clone XMG1.2, Biolegend/eBioscience).
Cd3 pe dazzle 594 clone 17a2
Manufactured by BioLegend
CD3 PE/Dazzle 594 (clone 17A2) is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen. CD3 is a cell surface receptor complex found on T cells and is involved in T cell activation and signaling. This product can be used for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 apc cy7 clone gk1, by BioLegend (2 mentions)
Golgiplug, by BD (2 mentions)
Mouse il 2, by BioLegend (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Ifnγ fitc clone xmg1, by BioLegend (2 mentions)
Mouse il 2, by Thermo Fisher Scientific (1 mentions)
Purified cd16 32 clone 93, by BioLegend (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Cd16 32 clone 93, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Perm buffer, by BD (1 mentions)
2 protocols using cd3 pe dazzle 594 clone 17a2
1
Murine Splenocyte Cytokine Profiling
2
Cytokine Production in Mouse Splenocytes
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Mouse splenocytes were incubated with 5ng/ml mouse IL-2 (Biolegend), 1µl BD Golgiplug (Becton Dickinson, Franklin Lakes, NJ) and 10µg peptide (linear peptide sequences: PA D630: KGVED /GE/K MEITT, NP 147: TYQRTRALV) in 96-well V-bottom plates at 37◦C, 5% CO2 for 5 h. Cells were then stained with Zombie Aqua Fixable Viability kit (Biolegend) for 30 min, washed once and subject to antibody staining for 30 min with the following panel: purified CD16/32 (clone 93); CD3 PE/Dazzle 594 (clone 17A2); CD4 APC/Cy7 (clone GK1.5); CD8α PerCP/Cy5.5 (clone53-6.7); CD8β PerCP/Cy5.5 (clone YTS 156.7.7, all from Biolegend). Cells were washed once before fixation in 4% formaldehyde, and permeabilization with perm buffer (BD Bioscience), followed by intracellular staining with IFNγ FITC (clone XMG1.2, Biolegend). Samples were acquired by flow cytometry on BD LSRFortessa and analyzed using Flowjo. Total cytokine production was calculated by subtracting non-specific background using unstained controls.
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