The following monoclonal antibodies were used to stain cells: anti-CD11b v450, F4/80 APCeFluor780, Ki67 eFluor660, CD19 PerCpCy5.5, Thy1.1 PeCy7 (eBiosciences), CD11c fluorescein isothiocyanate (FITC) or allophycocyanin (APC), CD24 BV711, CD45.2 v500, CD103 biotin, I-Ab phycoerythrin (PE), Ly6C PeCy7, PD-L1 APC, streptavidin PerCpCy5.5, CD4 APC-H7, CD8 v450, CD62L Alexa700, IFN-γ APC, Vβ3 PE (BD Biosciences), or CD64 PE (BioLegend). Exclusion of propidium iodide was used to gate live cells. Cells were enumerated using CountBright absolute counting beads (Thermo Fisher Scientific). Samples were acquired using the Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar).
Pd l1 apc
Manufactured by BD
Sourced in United States
PD-L1-APC is a fluorescently-labeled antibody that binds to the Programmed Death-Ligand 1 (PD-L1) protein. PD-L1 is a cell surface protein that plays a role in the regulation of the immune system. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of PD-L1-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Ifn γ apc, by BD (2 mentions)
Cd4 percp cy5, by BD (2 mentions)
Cd45 fitc, by BD (2 mentions)
Pd 1 apc, by BD (2 mentions)
Streptavidin percp cy5, by BD (1 mentions)
Cd62l alexa700, by BD (1 mentions)
Ki67 efluor660, by Thermo Fisher Scientific (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Cd4 apc h7, by BD (1 mentions)
Cd64 pe, by BioLegend (1 mentions)
Cd19 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd103 biotin, by BD (1 mentions)
F4 80 apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Thy1.1 pecy7, by Thermo Fisher Scientific (1 mentions)
Ly6c pe cy7, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Cd86 apc, by BD (1 mentions)
Cd73 pe, by Thermo Fisher Scientific (1 mentions)
8 protocols using pd l1 apc
1
Comprehensive Immune Cell Profiling by Flow Cytometry
2
Immunophenotypic Characterization of GB-MSCs
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The trypsinized GB-MSCs and their respective PBMCs were washed with PBS, centrifuged at 280 g for 10 minutes, counted, and brought to a concentration of 1.105 cells. GB-MSCs were tested for expression of the following surface markers: PD-L1-APC (BD Pharmingen, USA), CD73-PE, CD90-FITC, CD105-PerCP/Cy5-5 (eBioscience, USA), CD45-FITC/CD34-PE, CD44-FITC, CD146-PE, and HLA-A, B, C-FITC (Becton Dickinson, USA) and intracellular markers: Nestin-PE, Sox-2-PerCP, and GFAP-Alexa Fluor 488 (eBioscience, USA). For the detection of intracellularly expressed markers, the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA) was used following the manufacturer's instructions.
PBMCs from healthy volunteers were tested for surface expression of CD3-FITC, CD4-PerCP, CD161-PE, CD196-Alexa Fluor 488, CD25-FITC, CD14-FITC, CD80-PE, CD86-APC, and HLA-DR-PerCP and intracellular expression of FoxP3-PE (BD Pharmingen, USA) by using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA). Cells were processed according to the manufacturer's instructions, fixed with CellFix (BD, USA), and analyzed by FACSCalibur flow cytometer (BD, USA). Software CellQuest and WinMDI 2 were used for further analysis.
PBMCs from healthy volunteers were tested for surface expression of CD3-FITC, CD4-PerCP, CD161-PE, CD196-Alexa Fluor 488, CD25-FITC, CD14-FITC, CD80-PE, CD86-APC, and HLA-DR-PerCP and intracellular expression of FoxP3-PE (BD Pharmingen, USA) by using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA). Cells were processed according to the manufacturer's instructions, fixed with CellFix (BD, USA), and analyzed by FACSCalibur flow cytometer (BD, USA). Software CellQuest and WinMDI 2 were used for further analysis.
3
Multiparameter Flow Cytometry for Immune Profiling
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The following antibodies were used: antihuman CD3-FITC or V500, CD4 PerCP-Cy5.5, CD8 FITC or V450, CD56 PE or BV510, IFN-γ APC, PD-L1 APC, and mIgG1 APC isotype control (all from BD Biosciences). Cell surface staining was performed by incubating cells with mAbs for 30 min at 4°C in FACS staining buffer (BD Biosciences) followed by 2 times washing with phosphate-buffered saline. For intracellular staining, surface stained cells were fixed and permeabilized using fixation/permeabilization buffer (Thermo Fisher Scientific) and stained with anti-IFN-γ mAb according to the manufacturer’s instructions. Samples were resuspended in FACS buffer and acquired using a LSRFortessa flow cytometer (BD Biosciences) with FACSDiva software and analyzed using FlowJo software (FlowJo). For FACS data analysis, doublets were excluded using forward scatter height and width properties and dead cells were excluded by FVD780 (Thermo Fisher Scientific) positive staining.
4
Comprehensive Immune Cell Profiling
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The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich). Viability was assessed with Zombie Aqua (BioLegend). Samples were Fc receptor-blocked using mouse FcR blocking reagent (1:10; Miltinyi Biotec). For intracellular staining, samples were fixated and permeabilized with eBioscience Fixation/Permeabilization Concentrate, Diluent and 10X Buffer (Invitrogen). Data were acquired on FACSCanto II (BD Biosciences) or ACEA NovoCyte Quanteon (Agilent) and analyzed with FlowJo V.10.6.1 (Tree Star). Gating strategies can be found in online supplemental figures 8–13 .
5
Tumor Dissociation and Flow Cytometry Analysis
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Tumors were minced into small (1–2 mm3) pieces, and digested using a Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. The resulting supernatant was filtered through a 200-mesh sieve and washed twice with ice-cold PBS12 . healthy donors were incubated for 5 min with Fc-block (BD Biosciences). The phenotype of MDSCs in patients was determined by a combination of surface markers including HLADR-PerCP-Cy5.5 (Clone L243, BD Biosciences), CD33-FITC (Clone P67.6, BD Biosciences), CD11b-APC (Clone CBRM1/5, BD Biosciences), and CD45-BV421 (Clone HI30, BD Biosciences). Afterward, PD-L1 membrane expression was evaluated using PD-L1 (CD274)-PE (Clone 29E.2A3, BD Biosciences).
For the mouse model, single-cell suspensions were stained with either a myeloid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD11b-APC-Cy7 (Clone M1/70, BD Biosciences), Gr-1-PE (Clone RB6-8C5, BD Biosciences), and PD-L1-APC (Clone 10F.9G2, BD Biosciences) or a lymphoid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD4-PerCP/Cy5.5 (Clone RM4-5, BD Biosciences), CD8-PE (Clone 53–6.7, BD Biosciences), CD62L-APC (Clone MEL-14, BD Biosciences), and CD107a-APC (Clone 1D4B, BD Biosciences). Flow cytometry was performed with a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (TreeStar, Inc, Ashland, OR).
For the mouse model, single-cell suspensions were stained with either a myeloid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD11b-APC-Cy7 (Clone M1/70, BD Biosciences), Gr-1-PE (Clone RB6-8C5, BD Biosciences), and PD-L1-APC (Clone 10F.9G2, BD Biosciences) or a lymphoid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD4-PerCP/Cy5.5 (Clone RM4-5, BD Biosciences), CD8-PE (Clone 53–6.7, BD Biosciences), CD62L-APC (Clone MEL-14, BD Biosciences), and CD107a-APC (Clone 1D4B, BD Biosciences). Flow cytometry was performed with a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (TreeStar, Inc, Ashland, OR).
6
Comprehensive Immunophenotyping of Lung Cancer
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Flow cytometry was performed using fluorescence-conjugates or specific mAb and their controls followed by species-specific conjugate (Supplementary Table 2 ) using a FACS CantoII flow cytometer (Beckman Coulter) or a LSRFortessa (Becton Dickinson) from the flow cytometer facility Tuebingen. Positive cells in percentage (%) were calculated as follows: Surface expression in percent obtained with the specific antibody—surface expression in percent obtained with isotype control. Platelets were preselected by CD41a+ and CD62P− (resting) or CD62P+ (activated). Data analysis was performed using FlowJo software (v.10). In order to verify the reproducibility of our flow cytometry system, we performed a Bland–Altman analysis (Supplementary Fig. 8f ). For immunophenotyping of PBMC subsets of lung cancer patients and healthy control donors were identified by counterstaining with CD3-PECy5 (BD biosciences, San Diego, CA), CD19-APC/Fire750, CD4-Pacific Blue, CD8a-BV605, CD56-PECy7, CD14-BV785, HLA-DR-BV650 (Biolegend, San Diego, CA) and CD16-FITC (invitrogen). PD-1 and PDL-1 expression as well as activation levels were analyzed using a PD-1-APC or PDL-1-APC and a CD69-PE antibody (BD biosciences), respectively. Isotype controls were obtained from BD biosciences. Dead cells were excluded from analysis with LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific, Waltham, MA).
7
Enriching and Analyzing Tumor-Infiltrating Immune Cells
8
Immune Checkpoint Modulation in Liver Cancer
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Peripheral blood mononuclear cells (PBMC) were extracted from healthy volunteers by density gradient centrifugation in a lymphocyte separation medium (MP Biomedicals, Irvine, CA, USA). T cells in PBMC were activated and expanded with CD3, CD28 antibody, and 10 ng/ml IL-2 (Thermo Fisher Scienti c, Waltham, MA, USA), and then co-cultured with Hep3B cells at a ratio of 10:1 in the presence of a uorescent caspase 3 substrate (BD Biosciences, San Jose, CA, USA).
After 48h, PBMC and Hep3B were collected separately. The expression of immune checkpoints and apoptosis rate were measured by ow cytometry (BD FACSCelesta). All antibodies were purchased from BD Biosciences, including CD45-APC-CY7, CD3-PERCP-CY5.5, CD8-PE-CY7, PD1-APC, TIM3-BV650, LAG3-BV421, CTLA4-BV786, Caspase3-PE, and PDL1-APC antibodies.
After 48h, PBMC and Hep3B were collected separately. The expression of immune checkpoints and apoptosis rate were measured by ow cytometry (BD FACSCelesta). All antibodies were purchased from BD Biosciences, including CD45-APC-CY7, CD3-PERCP-CY5.5, CD8-PE-CY7, PD1-APC, TIM3-BV650, LAG3-BV421, CTLA4-BV786, Caspase3-PE, and PDL1-APC antibodies.
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