The following antibodies were used for staining: CD4 PerCp-Cy5.5, TCRβ PerCp-Cy5.5, and CD62L APC (TONBO); CD44 FITC, PD-1 (29F.1A12) PE-Cy7, CD69 PE-Cy7, CD5 PerCp-Cy5.5, CD8α Pacific Blue, and TCRβ Pacific Blue (BioLegend); CD44 PE-Cy7, CD8α (APC-eFluor 780), CD4 Qdot606, and Zap70 FITC (Life Technology). In vivo anti-PD (J43) antibody and control IgG were purchased from BioXcell. For intracellular flow cytometry, antibodies against phosphorylated ERKT202/Y204 (197G2), AKTThr308 (C31E5E), and ribosomal protein S6Ser235/236 (D57.2.2E) were purchased from Cell Signaling Technology.
Cd4 percp cy5
Manufactured by Cytek Biosciences
The CD4 PerCp-Cy5.5 is a fluorochrome-conjugated monoclonal antibody used for the detection and analysis of CD4 positive cells in flow cytometry applications. It is a reagent that binds specifically to the CD4 surface antigen expressed on T helper cells.
Automatically generated - may contain errors
Lab products found in correlation
Tcrβ percp cy5, by Cytek Biosciences (2 mentions)
Cd62l apc, by Cytek Biosciences (2 mentions)
Cd44 fitc, by Cytek Biosciences (2 mentions)
Cd69 pe cy7, by BioLegend (1 mentions)
Cd5 percp cy5, by BioLegend (1 mentions)
Cd8α pacific blue, by BioLegend (1 mentions)
Tcrβ pacific blue, by BioLegend (1 mentions)
Control igg, by BioXCell (1 mentions)
Cd25 percp cy5, by BioLegend (1 mentions)
Cd44 fitc, by BioLegend (1 mentions)
Pd 1 percp cy5, by BioLegend (1 mentions)
Cd4 buv395, by BD (1 mentions)
Cd8 buv737, by BD (1 mentions)
Cd8 pecy7, by Cytek Biosciences (1 mentions)
Cd69 pe, by Cytek Biosciences (1 mentions)
Cd86 pe, by Cytek Biosciences (1 mentions)
Cd11c apc cy7, by BioLegend (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
3 protocols using cd4 percp cy5
1
Multi-Marker Immune Cell Analysis
2
Multiparametric Flow Cytometry Analysis
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Single-cell suspensions from spleens, lymph nodes, and thymi were incubated with anti-CD16/32 at 5 µg/ml for 20 min on ice to block Fc receptors. Cells were stained with the following antibodies: CD4 PerCP-Cy55, TCRβ PerCp-Cy55, and CD62L APC (TONBO); CD44 FITC, PD-1 PerCP-Cy55, CD69 Cy7PE, CD25 PerCP-Cy55 (Biolegend); CD4 BUV395, and CD8 BUV737 (BD); Vα2 PE (phycoerythrin), Vα2 Pacific Blue, Vα2 FITC, FR4 Cy7PE, and CD73 BV605 (eBioscience); and CD25 BV605 (Life Technology). Dead cells were excluded using the live/dead fixable Near-IR death cell stain kit (Invitrogen). Intracellular Foxp3-FITC staining was done according to the manufacturer’s instructions (Life Technology). For detection of negatively selected thymocytes, caspase 3 PE (BD) was used as previously described (Breed et al., 2019 (link)). For intracellular flow cytometry, antibodies against phosphorylated ERKT202/Y204 (mAb 197G2), AKTS473 (Cell Signaling) were used as previously described (Hsu et al., 2009 (link)). Antibody against Alexa 647–anti-rabbit IgG was used as a secondary antibody to detect phosphorylation of ERK and AKT.
3
Multiparametric FACS Analysis of DC and T Cells
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FACS analysis was performed using standard methodology. Briefly, after Fc blocking, 1 × 106 cells were stained with appropriate antibodies for 30 min at 4°C and then washed with PBS/2% FBS. The cells were stained in FACS buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide) with fluorescently labelled antibodies from BioLegend (San Diego, CA; CD11c-APC/Cy7, CD11b-BV421, CD40-FITC, CD80-APC, CD86-PE, CD8-PE/Cy7, CD4-PerCp/Cy5.5, CD69-PE, CD25-APC, CD3-BV421, CD44-BD427, CD45.1-FITC, CD3-PerCp/Cy5.5, and CD44-FITC) or Tonbo Biosciences (San Diego, CA; HLA-DR-PerCp/Cy5.5) to identify DC and T cells. The percentage for each marker was determined by flow cytometry on a Sony SH800 cell sorter, and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Fluorescence minus one (FMO) control for each marker was used to identify gating boundaries. DC responses were examined by flow cytometry after staining with anti-CD11c, MHCII, CD8, CD4, CD11b, GR-1, CD103, CD80, and CD86 antibodies. TipDC were sorted using CD11bhi, Ly6c/GR-1hi, MHCII hi DC surface marker staining on a FACS Aria cell sorter (BD Biosciences, Franklin Lakes, NJ).
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