The monoclonal antibodies used in this study included CD3 PerCP, CD5 APC-R700, CD8-FITC, CD11c PE-Cy5, CD14 APC-H7, CD19 PE-Cy5, CD25 PE-Cy7, CD24 FITC, CD27 PE-Cy7, CD28 PE-Cy5, CD38 APC-H7, CD45 V500, CD45RO APC, CD56 APC, CD57 FITC, CD80 PE, CD86 APC, CD123 APC, CD127 BV421, CCR7 PE, TIGIT BV421, HLA DR V450, TIM3 PE, CXCR5 APC-R700, PD1 BV421, and PDL1 PE-Cy7 (BD Biosciences).
Pd 1 bv421
Manufactured by BD
Sourced in United States
PD-1-BV421 is a fluorescently-labeled anti-human PD-1 antibody that recognizes the programmed cell death-1 (PD-1) receptor. It is designed for use in flow cytometry applications to detect and quantify PD-1-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Ccr7 pe, by BD (3 mentions)
Cd25 pe cy7, by BD (2 mentions)
Cd27 pe cy7, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cd45ra apc, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Cd11c pe cy5, by BD (1 mentions)
Cd38 apc h7, by BD (1 mentions)
Cd127 bv421, by BD (1 mentions)
Tigit bv421, by BD (1 mentions)
Hla dr v450, by BD (1 mentions)
Tim 3 pe, by BD (1 mentions)
Pd l1 pe cy7, by BD (1 mentions)
Cd123 apc, by BD (1 mentions)
Cd14 apc h7, by BD (1 mentions)
Cd8 fitc, by BD (1 mentions)
Cd3 percp, by BD (1 mentions)
Cd24 fitc, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd86 apc, by BD (1 mentions)
8 protocols using pd 1 bv421
1
Comprehensive Immune Cell Profiling
2
Multicolor Flow Cytometry Panel for Immune Profiling
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One million live cells were counted using trypan blue and first stained with a fixable viability dye (eBiosciences 65-0866-14, Thermo Fisher Scientific; Waltham, MA, USA) for 30 min at 4 °C. Cells were washed in flow wash buffer (PBS with 0.05% sodium azide and 3% FBS) and stained with surface antibodies for 40 min at 4 °C. Cells were washed in a flow buffer and fixed in a flow buffer plus 4% PFA before filtering through 40-μm mesh strainer tubes (BD Biosciences; San Jose, CA, USA). Flow cytometry was performed on the BD Fortessa X20 cytometer, and the data were analyzed using FlowJo Version 10 (Becton, Dickinson & Co.; Franklin Lakes, NJ, USA). Flow cytometry antibodies used from BD Biosciences (San Jose, CA, USA) include: CD45-APC-R700 (565478), CD8-PE (553033), CD4-APC-H7 (5560181), Ly6G-BV605 (563005), Ly6C-FITC (561085), CD11b-APC (561690), PD-1-BV421 (562584), NK1.1-BV650 (564143), and MHCII-PerCPCy5.5 (562363). Flow cytometry antibodies used from eBiosciences (Thermo Fisher Scientific; Waltham, MA, USA) include: F4/80-Super Bright 780 (78-4801-82).
3
Multiparameter Flow Cytometry of CSF Immune Cells
4
Phenotypic Analysis of CD8+ T Cells in HLH
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Standard of care immune workup was performed at the Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratory of CHOA and at the diagnostic immunology laboratory at Cincinnati Children’s Hospital Medical Center. Immune investigations consisted of lymphocyte subset analysis, perforin and granzyme B expression, CD107a degranulation assay, plasma soluble interleukin-2 receptor (SIL2R) levels, NK cell activity, and cytotoxic T lymphocyte (CTL) function.
In a limited number of patients (three p-HLH and three T cell-EBV-HLH), additional T cell immunophenotyping for the purposes of research was performed. Peripheral blood mononuclear cells (PBMCs) were stained with CD3-PerCP/Cy5.5, CD8-BUV395, CD45RA-APC, CCR7-PE, HLA-DR-BV711, CD38-BUV496, and PD-1-BV421 antibodies (BD Biosciences and BioLegend). Live/dead fixable aqua dead cell stain (Thermo Fisher) was used to exclude dead cells in the analysis. Flow cytometry data was acquired on BD FACSymphony™ A5 and analyzed using FlowJo software v10. Effector memory (EM) population of CD8+ T cells was identified as CCR7− CD45RA− CD8+ T cells. Activated CD8+ T cells were identified as HLA-DR+CD38+ gated on CD8+ EM T cells or CD8+ T cells. Due to a lack of additional T-cell immunophenotypic data from infection-related HLH (iHLH) and MAS cohorts, our T cell activation analysis was restricted to p-HLH and T cell-EBV-HLH.
In a limited number of patients (three p-HLH and three T cell-EBV-HLH), additional T cell immunophenotyping for the purposes of research was performed. Peripheral blood mononuclear cells (PBMCs) were stained with CD3-PerCP/Cy5.5, CD8-BUV395, CD45RA-APC, CCR7-PE, HLA-DR-BV711, CD38-BUV496, and PD-1-BV421 antibodies (BD Biosciences and BioLegend). Live/dead fixable aqua dead cell stain (Thermo Fisher) was used to exclude dead cells in the analysis. Flow cytometry data was acquired on BD FACSymphony™ A5 and analyzed using FlowJo software v10. Effector memory (EM) population of CD8+ T cells was identified as CCR7− CD45RA− CD8+ T cells. Activated CD8+ T cells were identified as HLA-DR+CD38+ gated on CD8+ EM T cells or CD8+ T cells. Due to a lack of additional T-cell immunophenotypic data from infection-related HLH (iHLH) and MAS cohorts, our T cell activation analysis was restricted to p-HLH and T cell-EBV-HLH.
5
Immune Cell Profiling in HCC Patients
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Peripheral blood mononuclear cells were isolated through Ficoll (GE Healthcare Bio‐sciences AB, Sweden) from whole blood of patients with HCC and resuspended in 0.5 ml of PBS. Then the immunofluorescent antibodies were deployed to further distinguish the different kinds of immune cells in peripheral blood. The immunofluorescent antibodies used in the study were shown as followed: CD3‐FITC, CD4‐APC, CD25‐PE‐Cy7, PD‐1‐BV421, Foxp3‐V450, CTLA‐4(CD152)‐PE, TIM‐3‐PerCP‐Cy5.5, CD8‐ APC‐Cy7, CD56‐PE, and CD19‐ PerCP‐Cy5.5 (BD Bioscience). The related isotype antibodies were used as negative control. The result was analyzed with Flowjo 10.6.2 (BD Bioscience).
6
Phenotypic and Functional Characterization of CB CD19-CAR T Cells
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All anti-human antibodies, including CD45RA-APC (Cat: 550855), CD3-FITC (Cat: 555339), CD4-APC-Cy7 (Cat: 557871), CD8-PerPCy5.5 (Cat: 560662), CCR7-PE (Cat: 552176), CD27-PE-Cy7 (Cat: 560609), CD28-BV711 (Cat: 563131), Fixable Viability Stain (FVS) (Cat: 562247), PD-1-BV421 (Cat: 562584), and TIM-3-BV605 (Cat: 747961), were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA). Tumor cells were labeled with 2 µM intracellular tracing reagent carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Waltham, MA, USA), and dead cells were marked with FVS. All flow cytometric analyses were performed using a BD FACSCanto (BD Biosciences) and analyzed with FlowJo Version 10 (Tree Star, Ashland, OR, USA). The capacity of CB CD19-CAR T cells recognizing and killing target cells was evaluated by analyzing the percentage of CFSE-labelled target cells after coculturing for 24 h at different effector: target (E: T) ratios of 1:1, 2:1, 5:1, 10:1. Supernatants were harvested after 48 h, and multiple cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were detected using the BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit (BD Pharmingen) by flow cytometry.
7
Multiparametric Flow Cytometry Analysis
8
Multiparameter Phenotyping of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were incubated with directly conjugated mAbs for 30 minutes at 4 ℃. The cells were then washed before flow cytometric analysis. Monoclone Abs used were anti-human CD3-BV605, CD45-BV786, CD14-BV711, HLA-DR-Percp-Cy5.5, CD11b-AF700, Linage (CD19, CD20, CD56, CD40)-FITC, CD4-BV711, CD8 APC-H7, CD25-PE, CD127-BV510, PD-1-BV421, CD-45RA-AF700 (BD Biosciences), VISTA-AF647 (R&D Systems), CD33-APC-cy7 (eBioscience). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences) and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).
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