Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.
Anti cd3 pe cy7
Manufactured by BD
Sourced in United States
Anti-CD3-PE-Cy7 is a monoclonal antibody conjugate used for the detection and quantification of CD3-positive cells in flow cytometry applications. The antibody is labeled with the fluorescent dye PE-Cy7, which enables the identification and enumeration of T cells within a sample.
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Lab products found in correlation
Anti cd4 apc cy7, by BD (4 mentions)
Anti cd56 pe, by BD (3 mentions)
Anti cd4 horizon v500, by BD (3 mentions)
Anti cd4 pe, by BD (3 mentions)
Anti cd14 apc, by BD (3 mentions)
Anti ifn γ apc, by BD (3 mentions)
Anti cd16 pe cy7, by BD (3 mentions)
Anti cd4 percp cy5, by BD (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti cd62l apc, by BD (2 mentions)
Anti cd45.2 fitc, by BD (2 mentions)
Anti cd4 fitc, by BD (2 mentions)
Anti cd16 fitc, by BD (2 mentions)
Anti cd8 apc cy7, by BD (2 mentions)
Anti ifn γ fitc, by BD (2 mentions)
Anti cd31 fitc, by BD (2 mentions)
Anti cd8 apc, by BD (2 mentions)
Anti cd8 apc h7, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Anti cd57 apc, by BD (2 mentions)
37 protocols using anti cd3 pe cy7
1
Multiparametric Flow Cytometry Analysis
2
Lymph Node Immunophenotyping by Flow Cytometry
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The following extracellular, anti-human monoclonal antibodies were used for lymph node immunophenotyping: anti-CD3 PE-Cy 7, anti-CD8+PE-Cy 5, anti-CD4 FITC, anti-CD62L PE-Cy 5, anti-CD69 FITC, anti-CD152 (CTLA-4) PE, anti-CD11c PE-Cy 5, anti-CD86 PE-Cy 7, anti-HLA-DR PE, anti-CD3 FITC, anti-CD14 FITC, anti-CD16 FITC, anti-CD19 FITC, anti-CD123 PE-Cy 7, anti-CD141 PE, anti-CD68 PE-Cy 5, anti-CD20 PE, anti-CD56 PE, anti-CD279 (PD-1) APC, anti-CD11b PE, anti-CD64 FITC (BD Pharmingen, San Jose, CA).
The human monoclonal antibodies, anti-CD4 PE-Cy 5 and anti-CD25 PE, were purchased from Biolegend (San Diego, CA) and used in conjunction with intracellular staining with anti-FoxP3 FITC for the quantification of regulatory T cells. Cell death was determined by propidium iodine staining (eBioscience, San Diego, CA USA). Four-color flow cytometry was performed on a Guava 8HT flow cytometer (EMD Millipore, Billerica, MA USA) capturing 25,000 events for all samples. Results were analyzed using Guava Soft Incyte (EMD Millipore, Billerica, MA USA).
The human monoclonal antibodies, anti-CD4 PE-Cy 5 and anti-CD25 PE, were purchased from Biolegend (San Diego, CA) and used in conjunction with intracellular staining with anti-FoxP3 FITC for the quantification of regulatory T cells. Cell death was determined by propidium iodine staining (eBioscience, San Diego, CA USA). Four-color flow cytometry was performed on a Guava 8HT flow cytometer (EMD Millipore, Billerica, MA USA) capturing 25,000 events for all samples. Results were analyzed using Guava Soft Incyte (EMD Millipore, Billerica, MA USA).
3
Comprehensive Immune Profiling Workflow
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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.
4
Ex vivo Analysis of Cytokine Responses
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Unstimulated BAL cells were stained ex vivo with anti-CD3-PE-Cy7 and CCR5-PE (both from BD) and with CD4-PE-Cy5.5 and CD8-Qdot705 (both from Invitrogen). Freshly stimulated BAL cells and stimulated cryopreserved blood cells were washed and stained with anti-CD4-PE-Cy5.5 and CD8-Qdot705 (both from BD), permeablized, and stained intracellularly with CD3-APC-H7, IFN-γ-Alexa700, and interleukin 2 (IL-2)-APC (all from BD) and with tumor necrosis factor α (TNF-α)-PE-Cy7 (eBiosciences). Cells were acquired on a BD Fortessa, using FACSDiva software, and data were analyzed using FlowJo (TreeStar) and Pestle and Spice [25 (link)]. A positive cytokine response was defined as a level that was twice the background level, a net response of >0.05%, and an event cutoff of 10 events, and all data are reported after subtraction of the background level.
5
Intracellular Cytokine Profiling of Activated PBMCs
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PBMCs were stimulated with anti-CD3 antibody (0.1 μg/ml) for 6 h. After 1 h of incubation, brefeldin A and monensin (BD Biosciences) were added to stimulate intracellular cytokine protein accumulation. Following surface staining with anti-CD3-PE-Cy7, anti-CD4-Horizon V500, anti-CD8-APC-Cy7, anti-CD31-FITC, anti-CXCR4-PerCP-Cy5.5, and anti-CD28-APC-H7, the cells were fixed and permeabilized using the Fixation/Permeabilization Buffer Kit and further stained for intracellular cytokines with anti-TNF-α-FITC, anti-IFN-γ-FITC, anti-IL-6-PE, and anti-IL-10-APC (all from BD Biosciences).
6
Allogeneic Mixed Leukocyte Reaction Assay
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Allogeneic mixed leukocyte reactions (MLRs) were carried out as described previously (11 (link)). Briefly, naïve CFSE-stained T cells (positively selected on anti-CD3 magnetic beads) were added in a ratio of 10:1 to allogeneic human monocyte-derived DCs (1 × 104 cells/well in 96-well plates) that had been differentiated in the presence of conditioned HCT116 culture supernatants in X-Vivo full medium. After 5 days, cells were collected, T cells were stained with anti-CD3-PE-Cy7, anti-CD4-PE, and anti-CD8-APC (all from BD Biosciences), and T cell proliferation was determined by flow cytometry based on the decline in T cell CFSE fluorescence intensity. Results are displayed as x-fold proliferation values normalized on the values obtained with HCT116 control supernatants.
7
Immune Cell Analysis of COVID-19 Vaccinated Donors
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Human PBMCs collected from likely COVID-19-vaccinated donors between February 2022 and February 2023 were isolated from buffy coats by means of Ficoll density gradient centrifugation and infected for 24 h with 5 MOI of Prime-2-CoV_Beta (SPEk103) or left untreated. Cells were collected by scraping, washed in PBS and incubated for 30 min at 4 °C with the following antibody cocktail in PBS: anti-CD14 APC (BD Biosciences, San Jose, CA, USA, 561383), anti-CD4 BV605 (BD Biosciences, 562843), anti-CD8 APC-H7 (BD Biosciences, 561423), anti-CD19 BV786 (BD Biosciences, 563325), anti-CD3 PE-Cy7 (BD Biosciences, 557749), anti-CD56 PE (BD Biosciences, 556647), anti-CD25 BV421 (BD Biosciences, 567485), LIVE/DEAD™ Fixable Aqua stain (Invitrogen, Waltham, MA, USA, L34957). After three washes in PBS, cells were fixed in 4% formaldehyde for 20 min, washed and finally resuspended in FACS buffer. Data were acquired with an Attune NxT cytometer (Thermo Fisher Scientific). Data analysis was performed using Kaluza software (version 2.1, Beckman Coulter, Brea, CA, USA).
8
T Cell Activation and IFN-γ Production
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Fresh PBMC (2.5 × 106 cells/mL) were incubated with anti-CD28 (2 μg/mL) and anti-CD3 (1 μg/mL) for 18 hours, then Golgi stop (BD PharMingen) was added and incubated for an additional 6 hours. The cells were stained with anti-CD3-PE-cy7 and anti-CD4-APC-cy7, then permeabilized with Cytofix/Cytoperm (BD PharMingen) for 30 minutes and stained with anti-IFN-γ-APC (BD PharMingen). Cells were detected by flow cytometry.
9
Multicolor Flow Cytometry Panels
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The following flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA): anti-CD3-PE Cy7, anti-CD4-Alexa488, anti-CD8-PacBlu, anti-CD28-PE, anti-CD57-APC, anti-CD45RO-Alexa 700, anti-HLA-DR-APC, anti-CD14-PacBlu, anti-CD11a-FITC, anti-CD16-PE-Cy7, anti-CD163-PE, anti-CD62L-APC, and anti-CD86-Alexa 700. Two multi-color panels were used for T cells, and two panels were used for monocytes. Data were collected using a BD LSRII flow cytometer and analyzed with FCS Express software (DeNovo Software, Los Angeles, CA).
10
Immunophenotyping of Murine Splenocytes
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Cells used for immunophenotyping studies were either splenocytes or leukocytes obtained from blood. When mice were euthanized, blood was collected via cardiac puncture using a syringe that had been rinsed with 10% potassium ethylenediaminetetraacetic acid. The blood was placed in a 1 mL Wintrobe tube (Fisher Scientific, Hampton, New Hampshire) and centrifuged at 2000 rpm for 10 minutes. The plasma was removed and stored at −20°C for future analysis. The buffy coat was collected and placed in 2 mL of red blood cell solution for 10 minutes at room temperature. Then 10 mL of PBS was added and the tubes were centrifuged at 1500 rpm for 5 minutes. The cells present in the pellet or splenocytes were incubated with a panel of labeled monoclonal antibodies: anti-CD3 PE-Cy7, CD4 BV605, CD8 APC-H7, natural killer (NK)1.1 APC, and CD19 V450 (BD Biosciences, San Jose, California) in staining buffer on ice in the dark for 20 minutes. Cells were then washed in the staining buffer and resuspended in staining buffer and analyzed on an LSRII flow cytometer (BD Biosciences). Isotype controls were used for each experiment. Results were analyzed using FlowJo version 10 software (FlowJo, LLC, Ashland, Oregon).
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