Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5 . Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1 ).
Anti pd 1 percpcy5
Manufactured by BD
Sourced in United States
Anti-PD-1 PercpCy5.5 is a fluorescent-conjugated antibody that binds to the PD-1 (Programmed Death-1) receptor. It is designed for use in flow cytometry applications to identify and quantify PD-1-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 apc cy7, by BioLegend (1 mentions)
Anti cd8 amcyan, by BD (1 mentions)
Anti ccr7 fitc, by R&D Systems (1 mentions)
Live dead blue dye, by Thermo Fisher Scientific (1 mentions)
Anti ctla4 pe cy7, by BioLegend (1 mentions)
Anti cd244 fitc, by BioLegend (1 mentions)
Anti cd45ra af700, by BioLegend (1 mentions)
Anti cd160 pe, by BioLegend (1 mentions)
Facsaria 1, by BD (1 mentions)
Anti cd3 apc, by BD (1 mentions)
Anti cd57 apc, by BioLegend (1 mentions)
Violet amine reactive dye, by Thermo Fisher Scientific (1 mentions)
Anti cd3 petr, by Thermo Fisher Scientific (1 mentions)
Anti cd4 apc cy7, by BD (1 mentions)
Facslyric, by BD (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Anti cd8 apc, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Foxp3 transcription factor fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Anti cd3 v500, by BD (1 mentions)
3 protocols using anti pd 1 percpcy5
1
Detailed Immunophenotyping of T Cell Subsets
2
Immune Exhaustion and Senescence Analysis
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The expression of immune exhaustion (PD-1) and senescence (CD57) markers was assessed as described previously (26 (link)). Briefly, after revival and resting overnight, PBMCs were resuspended in 1 ml PBS and then stained with violet amine reactive dye (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature for differentiation of dead and live cells. The samples having more than 90% viability were considered for further analysis. The cells were washed again and incubated with a cocktail of antibodies [anti-Vα24 PE, anti-Vβ11 FITC (Beckman Coulter, Marseilles, France), anti-CD57 APC (Biolegend, USA), anti-CD3 PETR (Invitrogen, Carlsbad, CA, USA), anti-CD3 APC, and anti-PD1 PerCpCy5.5 (BD Biosciences)] for 30 min at room temperature. After washing, the cells were fixed in 3% paraformaldehyde, acquired on FACSAria-I (BD Biosciences, USA) and analyzed using FACSAria-I (BD Biosciences, USA) and analyzed using FACSDiva software version 6.1.3 (BD Biosciences, USA).
3
Multiparametric Phenotyping of PBMCs
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PMBCs were thawed in Benzonase Nuclease (Sigma-Aldrich)/X-VIVO medium (Lonza), washed with RPMI medium and stained with the following fluorochrome-conjugated antibodies: anti-CD14 – PerCP-Cy5.5 (BD, clone MΦP9), anti-HLA-DR – V500 (BD, clone G46–6), anti-PD-L1 – PE-Cy7 (BD, clone MIH1), anti-CD73 – BV605 (Biolegend, clone AD2), anti-CD3 – V500 (BD, clone SP34–2), anti-CD4 – APC-Cy7 (BD, clone RPA-T4), anti-CD8 – APC (BD, clone RPA-T8), anti-CD69 – PE-Cy7 (BD, clone FN50), anti-PD-1 – PerCP-Cy5.5 (BD, clone EH12.1) and anti-CD25 – BV421 (BD, clone M-A251). Dead cells were discriminated with the Fixable Viability Stain A×700(BD). To reduce unspecific antibody binding, FcR Blocking Reagent (Miltany) was added. Analysis of the intracellular marker FOXP3 – Alexa 488 (BD, clone 259D/C7) were conducted using the FOXP3/Transcription Factor Fixation/Permeabilization kit (ThermoFisher). Acquisition was performed by 10-color flow cytometry using BD FACSLyric with FACSuite software (BD Biosciences). FlowJo V 10 software (BD Biosciences) was used to analyze at least 106 events. Positively stained cells were gated according to the fluorescence minus one (FMO) control.
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