ELISA kits for murine IL-6 was from R&D Systems (Minneapolis, MN). Fluorescein-conjugated mAbs including anti-CD3-PE-Cy7, CD4-PE-Cy5, CD8-PE, CD8-FITC, CD11b-APC, Ly6G-PE, Ly6C-FITC, CXCR2-Percp-Cy5.5, CD45-BV510, PD1-PE, PDL1-PE, LAG3-PE, CTLA4-PE, IFNγ-PE and isotype antibodies were purchased from BD biosciences. TIM3-PE was from Miltenyi Biotec (Bergisch-Gladbach, Germany). Antibody against STAT3 (79D7, 4904S), p-STAT3 (Tyr705, 9131S), ZEB1 (3396P), ZO-1(5406P), Snail(3879P), N-Cadherin(4061P) were obtained from Cell Signaling Technology (Beverly, MA, USA) and antibody against Actin was from Santa Cruz. IL6 inhibitor (501109) and IFN-γ inhibitor (505827) was from Biolegend. Luciferin substrate (K9909PE) for in vivo image was purchased from PerkinElmer Inc (Hopkinton, MA, USA). HE staining kit was from Beyotime Biotechnology. ShRNA for ZEB1 (TG513177) was purchased from OriGene.
Ctla 4 pe
Manufactured by BD
Sourced in United States
CTLA-4-PE is a lab equipment product designed to detect and measure the presence of CTLA-4 (Cytotoxic T-Lymphocyte-Associated Protein 4) in biological samples. It uses a phycoerythrin (PE) fluorescent label to facilitate identification and quantification of CTLA-4 expression.
Automatically generated - may contain errors
Lab products found in correlation
Pd 1 pe, by BD (3 mentions)
Cd25 pe cy7, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Lag3 pe, by BD (2 mentions)
Cd8 pe, by BD (2 mentions)
Pd 1 apc, by BD (2 mentions)
I a 1 e, by BD (2 mentions)
Ex527, by Bio-Techne (2 mentions)
Apc cy7, by BioLegend (2 mentions)
Efluor 660, by Thermo Fisher Scientific (2 mentions)
Brilliant violet 421, by BioLegend (2 mentions)
Integrin α4β7, by BioLegend (2 mentions)
F4 80, by BioLegend (2 mentions)
Apc cy7, by BD (2 mentions)
Cd103, by BioLegend (2 mentions)
Cd8 pe cy7, by BioLegend (2 mentions)
Cd3 fitc, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Cd8 fitc, by BD (1 mentions)
18 protocols using ctla 4 pe
1
Comprehensive Immune Profiling Workflow
2
Comprehensive CD4+ T Cell Profiling
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One day post isolation FACS analysis was used to determine the percentages of various CD4+ T cell subsets, like naïve (TN), effector memory (TEM), central memory (TCM), T helper 17 cells (Th17), follicular helper T cells (Tfh) and regulatory T cells (Treg). For this the following antibodies were used: CD4-APC H7 (BD Biosciences, 560158), CD45RA-PE Cy7 (BD Biosciences, 560675), CD45RO-PerCP Cy5.5 (BD Biosciences, 560607), CCR7-APC (BioLegend, 353214), CCR6-PE (BD Biosciences, 559562), CXCR5-PerCP Cy5.5 (BioLegend, 335001), CD127-PE Cy7 (BD Biosciences, 560822), CD25-Horizon V450 (BD Biosciences, 560355). Further, the expression of different receptors on unstimulated as well as stimulated CD4+ T cells was determined using the following antibodies: CD4-APC H7, TCR-CD3-APC H7 (BD Biosciences, 560275), CD28-PerCP Cy5.5 (BD Biosciences, 560685), MHC-I-Horizon V450 (BD Biosciences, 561346), FAS-PE (BD Biosciences, 556641), FAS-L-PE (BioLegend, 306407), PD1-APC (BD Biosciences, 558694), PD1-L-PE Cy7 (BD Biosciences, 558017), TRAIL-PE (BD Biosciences, 550516), CTLA-4-PE (BD Biosciences, 555853), CD69-Horizon V450 (BD Biosciences, 560740), CD25-PE Cy7 (BD Biosciences, 557741), CXCR4-PerCP Cy5.5 (BD Biosciences, 560670) and CCR5-PE Cy7 (BD Biosciences, 557752). Cells were analyzed using the BD FACS Canto II with FACSDiva software.
3
Flow Cytometry Analysis of Murine Immune Cells
4
Comprehensive NK cell phenotyping
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PBMCs from subjects were resuspended in PBS buffer and were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed with 1× PBS before flow cytometry analysis. Antibodies used included anti-human CD3-BV786, CD19-BVAPC-H7, CD16-BV711, CD69 PE-CF594, CD57-BV421, PD-1-APC, CD70-PE, CD160-AF488, 2B4-AF700, CTLA-4-PE, TIM-3-FITC, 7-AAD (BD Biosciences, San Diego, CA, USA), CD56-BV510, BTLA-PE-CY7, CD39-APC (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7 (Ebioscience, San Diego, CA, USA), along with the corresponding isotype controls. NK cells were gated as CD3−CD14−CD19−CD56+CD16+, CD3−CD14−CD19−CD56+CD16−, or CD3−CD14−CD19−CD56−CD16+. Data were acquired with the LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.5 (Tree Star, Ashland, OR, USA).
5
Murine Immune Cell Staining Protocol
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The following antibodies were purchased from Biolegend or BD Bioscience: B220-Pacific Blue (RA3-6B2), CD3e-FITC & APC-Cy7 (145-2C11), CD4-APC (GK.1.5), CD8α-BUV395 & PE (53-6.7), CD11b-PerCP Cy5.5 (M1/70), CD11c-BUV786 (N418), CD25-APC-Cy7 (3C7), CTLA-4-PE (UC10-4F10-11), Gr-1-PE-Cy7 (RB6-8C5), ICOS-BUV510 (C398.4a), Ly6C-BUV510 (HK1.4), Ly6G-APC (1A8), MHC II (1-Ab)-FITC (KH74), MHC II (1-Ad)-FITC (39-10-8), and PD1-BUV786 (29F.1A12). αGalCer/mCD1d tetramers were provided by NIH tetramer facility.
6
Murine Lymphocyte Phenotyping by Flow Cytometry
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Hepatic mononuclear cells (HMNCs) and splenocytes were isolated as previously described [18 (link)]. Single-cell suspensions of HMNCs and splenocytes were labelled with fluorescent conjugated antibodies against mouse CD4-FITC, CD25-PE (eBioscience), CD62L-PE Cy7, CTLA-4-PE (BD Pharmingen), as well as CD103-PerCP-Cy5.5 (Biolegend). Nonspecific binding was blocked with staining buffer supplemented with 2% mouse serum and anti-CD16/anti-CD32. For intracellular staining of Foxp3, mouse regulatory T cell staining Kit (eBioscience) was used. Cells were labeled with surface antibody, fixed and permeabilized with freshly prepared Fixation/Permeabilization working solution according to the manufacturer’s instructions. After permeabilization, cells were further labeled with anti-mouse foxp3 (clone FJK-16s) (eBioscience). After incubation and resuspension, HMNCs and splenocytes were evaluated by flow cytometry, and the data were analyzed using Cell Quest software (Becton Dickinson).
7
Multiparametric Flow Cytometry Analysis
8
Phenotyping Cytotoxic T Cells via Flow Cytometry
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The antibodies used for CTL phenotyping (anti-CD3-ECD, CD4-PC5, CD8-PC7, PD-1-PE, CTLA-4PE, PD-L1 FITC; IgG1-FITC, IgG1-PE) were all obtained from BD Biosciences. To investigate competitive binding of staining antibody with blocking PD-1 mAb, flow cytometric analysis was performed using two different clones of staining antibody after 48 hrs: primary PD-1 (MIH4), secondary IgG1 FITC and PD-1 conjugated with PE.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
9
Comprehensive T-cell Immunophenotyping by Flow Cytometry
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At least 5 × 105 events per sample were acquired on a Gallios 10-color flow cytometer (Beckman Coulter). Phenotypic characterization of T-cell subsets was performed using CD3-APC-Cy7, CD39-APC, CTLA-4-PE (all BD Biosciences), CD4-PerCP/Cy5.5, CD8-PE/Cy7, PD-1-APC, PD-L1-PE/Cy7, CCR7-FITC, CD25-AF700, CD127-PerCP/Cy5.5, CD45RA-AF700 (all Biolegend), CD45-PE-eFluor610 (eBioscience) and aqua dead cell stain (Life Technologies).
10
Flow Cytometry Analysis of Murine Immune Cells
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For flow cytometry, we purchased mAbs to murine CD4 (Pacific Blue and APC-H7 from Biolegend, PE-CF594 and APC-Cy7 from BD Pharmingen), Ki-67 (PerCP-Cy5.5, clone B56, BD Pharmingen), Foxp3 (PE and eFluor450, clone FJK-16s, eBioscience), CTLA4 (PE, BD Pharmingen), I-A/I-E (FITC, BD Pharmingen), CD11b (APC from eBioscience, Pe/Cy7 from Biolegend), Ly-6G (APC, Biolegend), Ly-6C (Pacific Blue, Biolegend), Gr-1 (APC, Biolegend), F4/80 (APC and PE, Biolegend), CD11c (APC/Cy7, Biolegend), CD39 (eFluor 660, eBioscience), CD103 (PE, Biolegend), CD127 (Brilliant Violet421, Biolegend), CCR9 (FITC, Biolegend), and Integrin α4β7 (APC, Biolegend). For the analysis of mesenteric lymph nodes, we used the Aqua LIVE/DEAD® Fixable Dead Cell Stain Kit to exclude false positive signals from dead and apoptotic cells, and then washed two times prior to antibody staining. We purchased EX-527 from Tocris Bioscience. Drugs were dissolved in dimethylsulfoxide (DMSO), and DMSO was used as a control.
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