Thawed PBMC were washed and re-suspended, and the cell viability was measured by Trypan blue staining. A total of 2 × 106 cells were stained with anti-human CD4-FITC, anti-human CD25-PE, anti-human CD127-PE-Cy5, anti-human PD-1-APC and anti-human PD-L1-PE-Cy7 for surface antigens (eBioscience, San Diego, CA, USA), in accordance with the manufacturer’s instructions. For intracellular cytokine detection, 2 × 106 cells were stimulated with 2 μL of Cell Stimulation Cocktail and 2 μL of Protein Transport Inhibitor Cocktail (eBioscience, San Diego, CA, USA) for 5 h. Collected cells were first stained with fluorescein-labelled mAbs for surface antigens, followed by fixation using IC Fixation buffer (eBioscience, San Diego, CA, USA). Then, the cells were stained with anti-human IL-17A-PE (eBioscience, San Diego, CA, USA) in an appropriate volume of 1 × Permeabilization Buffer (eBioscience, San Diego, CA, USA) for 25 min. Finally, the cells were re-suspended in 300 μL of PBS for subsequent flow cytometric analysis. All data was acquired on FACScalibur (BD Biosciences, San Jose, CA), and processed using the CellQuest program (Becton Dickinson, Franklin Lakes, NJ).
Anti human pd 1 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-human PD-1-APC is a fluorochrome-conjugated antibody that binds to the human Programmed Cell Death 1 (PD-1) receptor. It is designed for use in flow cytometry applications to identify and characterize PD-1-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
Cellquest program, by BD (1 mentions)
Anti human il 17a pe, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Anti human cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti human cd25 pe, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Collagenase type 1, by Roche (1 mentions)
Paraformaldehyde (pfa), by Ted Pella (1 mentions)
Dispase 2, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Fk506, by Merck Group (1 mentions)
Gapdh g 9, by Santa Cruz Biotechnology (1 mentions)
Trypsin, by Biowest (1 mentions)
Cd25 pe, by Beckman Coulter (1 mentions)
Anti mouse cd45 apc, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti human pd 1 apc
1
Multiparametric Flow Cytometry of PBMC
2
Immune Cell Stimulation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For pharmacological stimulation, we used phorbol 12-myristate 13-acetate (PMA; Sigma) and ionomycin (Calbiochem). For antibody (Ab)-based stimulation, we used mouse antihuman or hamster antimouse monoclonal anti-CD3 and -CD28 Ab (555336, 555725, 553058, 553295; all from BD Pharmingen). For flow cytometry analyzes, we used LIVE/DEAD fixable red cell death staining kit (L23102, Invitrogen) and antimouse CD45-APC (L23102, eBioscience) or Percp-Cy5.5, CD8-BV610 and CD25-PCy7 (103132, 100744, and 102016, respectively; all from Biolegend), TCRβ-FITC (732247, Beckman Coulter) and PD-1-APC780 (47998582, eBioscience), antihuman PD-1-APC (17-9969-42, eBioscience) and CD25-PE (A07774, Beckman Coulter) and paraformaldehyde (TedPella). For western blot analysis, we used anti-DGKα (11 547–1-AP, ProteinTech), DGKζ (105195, Abcam), α tubulin (T5168, Sigma), GAPDH (G-9, Santa Cruz), horseradish peroxidase-conjugated antimouse and rabbit IgG (P0447, P0260, Dako), antirabbit IgG Dylight 800 (SA5-35571, Invitrogen) and mouse IgG AlexaFluor 680 (175775, AbcamLife). IKKβ inhibitor PS-1145 was from Sigma-Aldrich, MEK inhibitor PD98059 was from Calbiochem, and calcineurin (CaN) inhibitor FK506 was from Merck Millipore. Anti-PD-1 (nivolumab) humanized Ab was from BioVision. For TIL isolation, we used collagenase type I (Worthington), dispase II and DNase I (both from Roche). Trypsin was from Biowest.
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