According to the manufacturer’s instructions, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradients. PBMCs were stimulated with Leukocyte Activation Cocktail (BD Pharmingen, San Diego, CA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. PBMCs were stained for surface markers, fixed, permeabilized with IntraPreReagent (Beckman Coulter, Fullerton, CA), and then stained with antibodies for intracellular markers. Anti-human mAbs against APC-CD4, FITC-CD56, FITC-IFN-γ, PE-CF594-CD3, PE-IL-2, PE-TNF-α, and V450-CD8, with controls, were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA) and analyzed with FlowJo software (Ashland, OR).
Cd3 pe cf594
Manufactured by BD
Sourced in United States
The CD3 PE-CF594 is a fluorescently-labeled monoclonal antibody that binds to the CD3 antigen, which is present on the surface of T cells. It is designed for use in flow cytometry applications to identify and quantify T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BD (3 mentions)
Cd4 fitc, by BioLegend (3 mentions)
Gallios instrument, by Beckman Coulter (2 mentions)
Cd3 bv650, by BioLegend (2 mentions)
Diva software, by BD (2 mentions)
Cd19 pe cf594, by BD (2 mentions)
Cd49b pe cf594, by BD (2 mentions)
Cd11c bv650, by BioLegend (2 mentions)
F4 80 alexafluor 700, by BioLegend (2 mentions)
Cd45 bv605, by BioLegend (2 mentions)
Cd8 apc fire, by BioLegend (2 mentions)
Cd45 v500, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Ifn γ pe cy7, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Tnf α pe, by BD (2 mentions)
Il 2 apc, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Cd8 v500, by BD (2 mentions)
18 protocols using cd3 pe cf594
1
Multiparametric Flow Cytometry Analysis
2
Multiparameter Characterization of NK Cells
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PBMCs or tissue-infiltrating lymphocytes were stained as follows. Frozen cells were thawed and resuspended in complete RPMI1640 medium (Corning, 10–040-CVR) containing 10% fetal bovine serum (Gibco, 12662029), 1% glutamine (Immundiagnostic, K7732) and 1% penicillin and streptomycin (Gbico, 15140–122). 1×106 cells were used for each panel and stained in dark at room temperature (20–25 °C) for 30 min. The following monoclonal antibodies (Abs) were used for NK cell phenotypic characterization: Pacific Blue-Vivid (Invitrogen, CA), PE-CF594-CD3 (BD Biosciences, 562280), Alexa Fluor® 700-CD14 (BD Biosciences, 557923), PE-Cy7-CD56 (BD Biosciences, 335791), APC-NKG2D (BD Biosciences, 558071), PE-NKG2C (R&D, FAB138C), PE-NKG2A (R&D, FAB1059C), APC-CD69 (BD Biosciences, 340560), PerCP-Cy5-HLA-DR (BD Biosciences, 347364) and FITC-CD38 (BD Biosciences, 555459). After staining, the PBMCs were washed twice with phosphate buffer saline and detected using a BD LSR II Fortessa flow cytometer (BD Biosciences, NJ). The data were analyzed using the FlowJo software (TreeStar, San Carlos, CA).
3
Profiling Immune Cell Phenotypes and Functions
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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradients. For the phenotypic analysis, PBMCs were stained with FITC-LAG-3, FITC-CD8, PE-2B4, PE-CD8, PE-CTLA4, PE-CF594-CD3, PE-CY7-CD4, BV421-PD-1, V450-CD8 (BD Biosciences, Franklin Lakes, NJ), and APC-Tim-3 (eBioscience, San Diego, CA, USA). For cytokine analysis, PBMCs were stimulated with a Leukocyte Activation Cocktail (eBioscience, San Diego, CA, USA), at 37° C for 4 h prior to intracellular staining using Pharmingen's staining protocol. Anti-human monoclonal antibodies against FITC-IFN-γ, PE-TNF-α (eBioscience), and PB-IL-2 (Biolegend, San Diego, CA, USA) were used. Corresponding isotype-matched controls were purchased from BD Biosciences and eBioscience. Data was acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (Ashland, OR, USA). The gating strategy for cytokines and exhaustion markers is shown in Supplementary Figure 1 .
4
Comprehensive Tumor Immune Profiling
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Tumor cells isolated from mice were thawed and stained with LIVE/DEAD Fixable Violet Dead Staining Kit (ThermoFisher). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD45 BV605, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C PE, Ly6G APC-Cy7, MHC II FITC, CD80 PE-Cy7 (all from Biolegend) for myeloid cell identification and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, CD25 PE, CD44 PE-Cy7, CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using FACSFortessa flow cytometer with Diva software (Becton Dickinson).
5
Tumor-Targeted IL-10 Silencing in Mice
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Eight to ten-week old female C57BL/6 mice were subcutaneously inoculated in the right flank with MC38/0 cells (1.1 × 106/0.2 ml/mouse). On the 14th, 15th and 17th day of the experiment, mice were injected i.t. with LVs encoding shRNA against IL-10 (shIL10–3, 2x106TU/50 μl/mouse) or reference LVs encoding scrambled shRNA against human GAPDH (shN). Two days after the third injection, the mice were sacrificed and their tumor nodules were dissected and homogenized. Efficacy of transduction in tumors was measured by flow cytometry as the fluorescence intensity of EGFP among cells isolated from tumors. Concentration of IL-10 was estimated by ELISA in supernatants collected from 24 h culture of 5 mg tumor tissue/ml. Myeloid and lymphocyte populations in tumors were analyzed using LSR Fortessa with Diva software (Becton Dickinson) after staining with fluorochrome-conjugated antibodies: CD45 V500, CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C BV510, Ly6G BV605, MHC II APC-Cy7, for myeloid cell identification (all from Biolegend) and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, (all from BioLegend) for lymphocyte identification.
6
Phenotypic and Functional Analysis of Antigen-Specific T Cells
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To analyze the magnitude and phenotype of the HIV-1-, FLAG- or VACV-specific T cell immune responses, 2 × 106 splenocytes (erythrocyte-depleted) seeded on 96-well plates were stimulated for 6 h in complete Roswell Park Memorial Institute (RPMI) 1640 medium (100 units/mL of penicillin/100 μg/mL of streptomycin, 2 mM L-glutamine, 10 mM Hepes and 0.01 mM β-mercaptoethanol) with 10% FCS, anti-CD107a-FITC (BD Biosciences), 1 µL/mL Golgiplug (BD Biosciences), monensin 1X (Invitrogen) and 5 µg/mL of the different HIV-1 clade B consensus peptide pools or 5 µg/mL of FLAG peptide or 10 µg/mL of VACV E3 peptide. After stimulation, splenocytes from immunized mice were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and stained intracellularly with the following fluorochrome-conjugated antibodies: IL-2-APC, IFN-γ-PeCy7 and TNF-α-PE for functional analyses and CD3-PECF594, CD4-APCCy7, CD8-V500, CD127-PerCPCy5.5 and CD62L-Alexa700 for phenotypic analyses (all from BD Biosciences). The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).
7
Activation Profiling of T-cell Subsets
8
Characterization of Activated T Cells in PBMCs
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Cryopreserved peripheral blood mononuclear cells (PBMCs) from a subset of subjects from whom PBMCs were available were thawed and rested overnight at 37 °C in order to minimize the effect of cellular activation due to thawing. All antibodies were pre-titrated in order to determine appropriate working concentrations. Rested PBMCs were then stained with the following antibodies: aqua viability dye (Invitrogen), CD3-PECF594 (BD; Franklin Lakes, NJ; clone UCHT1), CD4-PerCP-Cy5.5 (Biolegend; San Diego, CA; clone RPA-T4), CD8-APC-H7 (BD, clone SK1), CD38-PECy7 (BD, clone HIT2), and HLA-DR-eFluor605 (eBioscience; San Diego, CA; clone LN3). All stains were performed at 4 °C, and cells were fixed with 4% paraformaldehyde after staining. Stained PBMCs were subsequently analyzed on an LSR Fortessa cytometer (BD), using Rainbow Fluorescent Particles (BD) and application settings in BDFACSDiva7 to correct for day-to-day variations in instrument performance. Data were analyzed in FlowJo10 (Treestar; Ashland, OR). Cells were gated on aqua viability dye negative (live), lymphocytes determined by forward scatter vs. side scatter, and subsequently gated on CD3+CD4 + or CD3+CD8 + cells. Levels of activation markers (CD38 and HLA-DR) were measured by percentage positivity on CD4 + and CD8 + cells based on gates created using fluorescence minus one (FMO) controls.
9
Multiparametric Characterization of Immune Cells from Cryopreserved Samples
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Cryopreserved PBMC/cord BMC (CBMC) were thawed, counted, and processed immediately for phenotypic assessment using two staining panels. Panel A consisted of surface staining with Zombie yellow (viability), CD25 FITC (BioLegend), Lag3 PE (Invitrogen), CTLA4 PE-CF594 (BD Biosciences), CD4 PerCP-Cy5.5 (BD Biosciences), CD3 Ax700 (BD Biosciences) followed by fixation and permeabilization using the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (eBioscience). Intracellular staining was then performed with FoxP3 Ax647 (BD Biosciences), Granzyme B APC-fire750 (BioLegend), IL-10 BV421 (BioLegend) and TGFβ PE-Cy7 (BioLegend). Panel B consisted of surface staining with Zombie yellow (viability), CD4 FITC (BioLegend), CD3 PE-CF594 (BD Biosciences), GITR PerCP-Cy5.5 (BioLegend), TNFR2 PE-Cy7 (BioLegend), CD39 Ax700 (R&D Systems), PD1 APC-Cy7 (BioLegend) and TIGIT BV421 (BioLegend). Intracellular staining consisted of FoxP3 Ax647 (BD Biosciences) and IL-35 PE (BioLegend). Analysis was performed using the Galios instrument (Beckman Coulter).
10
SARS-CoV-2-Specific T Cell Profiling
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To determine the magnitude and phenotype of the SARS-CoV-2-specific T cell responses, 4 × 106 splenocytes or 2 × 106 lung-derived lymphocytes (both cell types were erythrocyte-depleted) seeded on 96-well plates were stimulated ex vivo for 6 h in complete RPMI 1640 medium with 10% FCS, 1 µL/mL Golgiplug (BD Biosciences), anti-CD107a-FITC (BD Biosciences), monensin 1X (Invitrogen) and 1 µg/mL of the different SARS-CoV-2 peptide pools representing the S, M and N antigens (JPT Peptide Technologies GmbH, Berlin, Germany). After stimulation, lymphocytes were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and intracellularly stained by incubation with the following fluorochrome-conjugated antibodies: IFN-γ-PeCy7, IL-2-APC and TNF-α-PE for functional analyses and CD3-PE-CF594, CD4-APC-Cy7 and CD8-V500 for phenotypic analyses (all from BD Biosciences). Dead cells were excluded from the analysis using the LIVE/DEAD Fixable Violet Dead Cell Stain kit (Invitrogen). Cells were acquired in a GALLIOS flow cytometer (Beckman Coulter), and data analyses were performed using FlowJo software (Version 10.4.2; Tree Star). Lymphocyte-gated events ranged between 105 and 5 × 105. Background responses obtained in unstimulated controls (RPMI) were subtracted from the responses detected in stimulated samples.
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