Whole blood samples (100 μl) were collected from each child and stained with antibodies for the flow cytometry analysis. The antibodies used for the lymphocyte subset analysis included FITC-anti-CD3, PE-Cy7-anti-CD19, PE-anti-CD (16 + 56), PerCP-Cy5.5-anti-CD45, APC-Cy7-anti-CD4, Alexa Fluor 750-anti-CD8 (Beckman). For the flow cytometry analysis, surface and intracellular labeling were also performed. The antibodies used for T-cell phenotypes and function analysis included FITC-anti-CD43, PE-anti-CTLA-4, PE-anti-CD44, PE-Cy7-anti-CD62L, and FITC-anti-PD-1 (Biolegend). All cell suspensions were incubated for 20 min at room temperature. Red blood cells were then lysed with the lysing solution, and the cells were then washed and re-suspended in 200ul of PBS. The cells were collected by using the Beckman DxFLEX Flow Cytometer. Data obtained were analyzed with the Kaluza Analysis Software (Beckman Coulter). Based on the scatter signals and with the use of Fixable Viability Dye eFluor 780 (Invitrogen), cell debris and dead cells were removed from the analyses.
Pe cy7 anti cd62l
Manufactured by BioLegend
Sourced in United States
PE-Cy7-anti-CD62L is a fluorescently labeled antibody that binds to the CD62L protein, also known as L-selectin. This protein is expressed on the surface of various immune cells, including lymphocytes. The PE-Cy7 fluorescent dye is used to label the antibody, allowing for detection and analysis of CD62L-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Pe anti ctla 4, by BioLegend (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Anti cd44 pe, by BioLegend (1 mentions)
Anti cd43 fitc, by BioLegend (1 mentions)
Anti pd 1 fitc, by BioLegend (1 mentions)
Anti cd3 fitc, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Dxflex flow cytometer, by Beckman Coulter (1 mentions)
Anti cd44 bv510, by BD (1 mentions)
Facsymphony a5, by BD (1 mentions)
Percp anti cd43, by BioLegend (1 mentions)
Anti cd62l pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti cd44 bv510, by BioLegend (1 mentions)
Bv421 anti cd8, by BioLegend (1 mentions)
Anti cd11b fitc, by BioLegend (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Brilliant violet 510 anti cd8a, by BioLegend (1 mentions)
Apc cy7 anti tcrβ, by BioLegend (1 mentions)
Anti cd4 pe, by BioLegend (1 mentions)
3 protocols using pe cy7 anti cd62l
1
Lymphocyte Subset Analysis by Flow Cytometry
2
Quantifying Antigen-Specific CD8+ T Cells
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For H2-Kb Ova257-264 (SIINFEKL)-specific CD8+ T-cell analysis, spleen cells were obtained 12 days after tumor inoculation and stained with AF647-coupled dextramer loaded with Ova257-264 peptide (DexOT–I; Immudex, Copenhagen, Denmark), FITC-anti-CD11b, PerCP-anti-CD43, BV421-anti-CD8 (all from BioLegend, San Diego, CA, USA), Fixable Viability Dye eFluor 780, PE-Cy7-anti-CD62L (eBioscience), and BV510-anti-CD44 (Becton-Dickinson).
The frequency of CD8+ T cells specific for the H2-Db-restricted Leader-Gag-derived epitope GagL85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetIGagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown inSupplementary Figure 2
The frequency of CD8+ T cells specific for the H2-Db-restricted Leader-Gag-derived epitope GagL85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetIGagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown in
3
Multiparametric Flow Cytometry Analysis
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Cell suspensions were incubated with anti-mouse CD16/CD32 (Biolegend, 101,319). For cell surface flow cytometry, cells from spleens, thymuses and LNs were stained with specific antibodies for surface antigens as follows: PE-anti-CD4 (Biolegend, 100,408), Pacific Blue-anti-CD4 (Biolegend, 100,531), Brilliant Violet 510-anti-CD8a (Biolegend, 100,751), APC/Cy7-anti-TCRβ (Biolegend, 109,220), 7-AAD (BD Pharmingen™, 559,925), APC-anti-CD25 (Biolegend, 102,012), PerCP/Cy5.5-anti-CD44 (Biolegend, 103,032), PE/Cy7-anti-CD62L (Biolegend, 104,418), PE-anti-CD278 (ICOS) (Biolegend, 107,705), APC-anti-CD304 (Neuropilin-1, Nrp1) (Biolegend, 145,206), PE/Cy7-anti-CD279 (PD-1) (Biolegend, 109,110). For intracellular staining, cells were fixed and permeabilized with Fixation/Permeabilization Kit (eBioscience, 00–5123, 00–5223), washed with Permeabilization Buffer (eBioscience, 00–8333) and stained with PE-Cy7-anti-ki67 (eBioscience, 25-5698-82), PE-anti-CTLA4 (Biolegend, 106,306), AF488-anti-Foxp3 (Thermo Scientific™, 53-5773-82), PE-anti-Foxp3 (Biolegend, 126,404). For apoptosis analysis, cells were stained with FITC-AnnexinV (Biolegend, 640,906) in AnnexinV Binding Buffer (Biolegend, 422,201). Samples were analyzed by LSRII multicolor flow cytometer (BD Biosciences CA, USA) and data analysis was performed using the FlowJo software (Tree Star, USA).
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