For Tregs, PBMCS were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher) washed, and then stained with anti-CD4 AF700 and anti-CD25 PE-Cy7 (BD Biosciences). Cells were washed and fixed and permeabilized using a FoxP3 Fix/Perm Kit (eBioscience) and intracellularly stained with anti-CD3-Pacific Blue (BD Biosciences) and anti- FoxP3 FITC (eBioscience). For Th-17 cells, 106 PBMCs were rested overnight and then stimulated with PMA (75 ng/ml) and ionomycin (1 μg/ml, Sigma-Aldrich) and incubated for 1 hour before addition of GolgiStop (BD Biosciences) and brefeldin A (1 μg/ml, Sigma-Aldrich) for an additional 5-hour incubation. Cells were washed and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain. Cells were then washed and stained with anti-CD4 AF700 and anti-CD8 APC/H7 (BD Biosciences). Cells were washed and then fixed and permeabilized using BD Cytofix/Cytoperm Kits then stained with anti-IFN-γ PE-Cy7 and anti-CD3 Pacific Blue (BD Biosciences), and IL-17 AF647 (eBioscience). Samples were acquired on a BD LSR II. A minimum of 200,000 events were recorded for each sample. Data were analyzed using FlowJo software (Treestar).
Anti cd3 pacific blue
Manufactured by BD
Sourced in United States
Anti-CD3-Pacific Blue is a flow cytometry reagent used to detect and quantify T cells in cell samples. It is a monoclonal antibody labeled with the Pacific Blue fluorescent dye that binds specifically to the CD3 antigen expressed on the surface of T lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Golgistop, by BD (4 mentions)
Anti ifn γ fitc, by BD (4 mentions)
Anti mip1β pe, by BD (4 mentions)
Brefeldin a, by Merck Group (3 mentions)
Anti cd19 apc, by BD (3 mentions)
Anti cd8 apc cy7, by BD (3 mentions)
Anti cd16 apc cy7, by BD (3 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (3 mentions)
Rosettesep nk cell enrichment kit, by STEMCELL (3 mentions)
Anti cd56 pe cy7, by BD (3 mentions)
Anti ifn γ pe cy7, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Anti cd8 v500, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti cd19 apc cy7, by BD (2 mentions)
Anti cd8 alexa 700, by BD (2 mentions)
Anti cd4 percp cy5, by BD (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Anti cd38 apc, by BD (2 mentions)
33 protocols using anti cd3 pacific blue
1
Multicolor Flow Cytometry for Tregs and Th17
2
Detailed Immune Cell Phenotyping
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After homogenization of spleen tissue, immune cells were purified using 35% Percoll (GE Healthcare, La Chapelle-sur-Erdre, France) and red blood cells were lyzed. 106 leucocytes were incubated with anti-CD16/32 (BD Pharmingen, Rungis, France) to block non-specific binding and washed. Cells were then incubated 30 min with appropriate dilutions of anti-CD3-Pacific Blue, anti-CD8-APC-Cy7, anti-CD4-PE, anti-NK1.1-Percp-Cy5.5 and anti-CD19-APC antibodies, all purchased from BD Pharmingen. The staining of ST2 was assessed with a rat monoclonal anti-mouse ST2-FITC antibody (clone DJ8; MB Bioproducts). Cells were washed, fixed in PBS containing 2% FCS, 0.01 M sodium azide and 2% formaldehyde and analyzed on a FACS Aria II ® flow cytometer using BD FACS Diva software (BD Biosciences). Dead cells and doublet cells were excluded on the basis of forward and side scatter. The different immune cell types were identified and gated as follows: BL were CD19+; NK cells were NK1.1+/CD3−; T CD8+ lymphocytes were NK1.1−/CD3+/CD8+/CD4−; and T CD4+ lymphocytes were NK1.1−/CD3+/CD4+/CD8−. The gating strategy was previously described [5 (link), 12 (link)].
3
Multicolor Flow Cytometry Immunophenotyping
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Peripheral blood lymphocytes were immunophenotyped by multicolour flow cytometry using the following antibodies: anti-CD3 Pacific Blue, anti-CD56 FITC, Anti-CD27 PE, anti-CD28 APC, anti-CD1d PE, anti-CD19 APC, anti-CD27 V450, anti-CD38 PerCP-Cy5.5 (all from BD Biosciences, San Jose, CA, USA) and anti-CD45RA PerCP-Cy5.5, anti-CD62L PE-Cy7, anti-CD5 PE-Cy7, anti-CD24 APC-eFluor 780 and anti-CD4 APC-eFluor 780 (from eBioscience, Inc. San Diego, CA, USA). Staining was performed on whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturer’s instructions. A minimum of 250,000 events were acquired for T cell panels and 500,000 events for B cell panels to ensure adequate capture of rare populations. Subsequent detailed analysis of lymphocyte sub-populations was performed on the gated lymphocyte population using FlowJo (Treestar, Inc., OR, USA). Absolute counts for the different lymphocyte populations were calculated per litre of blood, based on haematology laboratory reported total lymphocyte count.
4
Phenotyping of Activated PBMC
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Freshly isolated PBMC at 5 × 105 cells/well were stimulated with anti-CD28 (0·5 ug/ml), peptide pools (ESAT-6, CFP-10, ESAT-6/CFP-10 (E6C10) or AIM alone for 6 hours at 37°C in 5% CO2 incubator. Cells were harvested after 14–18 h rest at 4°C, erythrocyte lysed, and then surface-stained in the dark. The following directly conjugated monoclonal antibodies were used: anti-CD3-Pacific Blue, anti-CD4-AmCyan, anti-CD25-Per-CP-Cy5·5, anti-CD161-PE, anti-CD39-APC, anti-CD147-FITC (BD Bioscience) and anti-CD127 PE-Cy7 (eBioscience).
5
Phenotypic Characterization of Immune Cells
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The following monoclonal antibodies (MAbs) were used for phenotypic analysis: (i) anti-CD27-APC-eFluor780, anti-CD45RA-phycoerythrin (PE), and anti-CD127-eFluor450 (eBioscience, San Diego, CA); (ii) anti-CD3-Pacific Blue, anti-CD8-AmCyan, anti-CD8-V500, anti-CD11a-fluorescein isothiocyanate (FITC), anti-CD95-PE, anti-Ki67-FITC, and anti-CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany); (iii) anti-CCR7-FITC (R&D Systems); and (iv) anti-PD-1-PE-Cy7 (BioLegend, San Diego, CA). 7-Aminoactinomycin D (7-AAD; Viaprobe; BD Biosciences) was used for the exclusion of dead cells. All samples were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
6
NK Cell Functional Assay with Immune Complexes
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Human NK cells were isolated from buffy coats using RosetteSep NK cell enrichment kit (StemCell Technologies) and Ficoll separation. The isolated NK cells were rested overnight at 1.5 × 106 cells/mL in IL-15 at 37°C. ELISA plates were coated with antigen at 300 ng/well and incubated for 2 hours at 37°C. Plates were blocked with 5% BSA in PBS overnight at 4°C. The next day, 100 μL of antibodies, at a concentration of 5 μg/mL, were added to the plates. Plates were incubated for 2 hours at 37°C to form immune complexes. After the incubation, NK cells were added to the plates at 5 × 104 cells/well in R10 supplemented with anti-CD107a PE-Cy5, Brefeldin A (MilliporeSigma, B7651-5MG), and GolgiStop (BD Biosciences, 555802). Plates were incubated for 5 hours at 37°C. Following the incubation, NK cells were stained for the surface markers with anti-CD56 PE-Cy7, anti-CD16 APC-Cy7, and anti-CD3 Pacific Blue (BD Biosciences, 557747, 557758, 558124). NK cells were fixed and permeabilized with Fix&Perm cell permeabilization kit (Invitrogen, Thermo Fisher Scientific). Cells were incubated with anti–MIP-1β PE and anti–IFN-γ FITC (BD Biosciences, 550078, 340449) to stain for intracellular markers. Cells were acquired on an Intellicyt iQue.
7
Phenotypic Characterization of Peritoneal Cells during Toxoplasma Infection
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On the third day of infection, peritoneal cells from WT mice infected with 1 × 107 Nc-1 tachyzoites were collected and stained for phenotypic characterization and Dectin-1 expression. Briefly, mice were euthanized, and their peritoneal cavities were washed with ice-cold PBS. The suspension was then centrifuged at 400 × g, at 4°C, for 10 min. The cell pellet was resuspended in PBS with 5% of normal rabbit serum, at room temperature, for 15 min, prior to incubation with the appropriate antibodies: anti-CD11b-APC-Cy7, anti-CD11c-FITC, anti-MHCII-PE, anti-CD11c-V450, anti-CD19-APC-Cy7, anti-CD3-Pacific Blue, anti-CD49b-APC (BD Biosciences), and anti-Dectin-1-PE (R&D Systems, Minneapolis, MN, USA). Cells were incubated with primary antibodies conjugated to the different fluorochromes for another 30 min, at room temperature. After washing, cells were suspended in PBS with 3% formaldehyde, read by flow cytometry (FACSCantoII, BD Biosciences), and analyzed using dedicated software (FlowJo X, Tree Star Inc., Ashland, OR, USA).
8
Quantifying CD38 Expression on CD8+ T Cells During HIV-1 Infection
9
CD8 T Cell-Dendritic Cell Interaction
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Epitope-specific CD8 T cells were added directly to autologous iDC at 3:1 effector to target (E/T) ratio in the presence or absence of peptide of interest (10 μM) and co-cultured for 48 hours. Immature dendritic cells cultured in the presence of a maturation cocktail containing TNFα (50ng/ml), IFNα (3000U/ml), IFNγ (1000U/ml), IL-1B (25ng/ml), and pI:C (20ug/ml) were used as a positive control. After two days, cells were stained with dead cell dye (Invitrogen), anti-CD3-Pacific Blue, anti-CD8-V500, anti-CD14-alexa700, anti-CD83-PE, and anti-CD86-FITC (all from BD Pharmingen) at 4°C for 30 min. Cells were then washed and events were acquired on an LSR II flow cytometer.
10
Phosflow and IL-6R Phenotype Analysis
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The following antibodies were used for Phosflow and IL-6R phenotype analysis in samples processed at Newcastle: anti-CD3-Pacific blue (UCHT1), anti-CD19-FITC (4G7), anti-CD19-APC (HIB19), anti-Stat3 (pY705)-Alexa Fluor 647 (4/P-STAT3) and anti-Stat1 (pY701)-Alexa Fluor 647 (4a; all BD Biosciences, Oxford, UK); anti-CD4-APC-eFluor 780 (SK3; eBioscience, Hatfield, UK); IL-6R-Fluorescein (17506; R&D Systems Europe, Abingdon, UK). Phosflow was performed on whole blood, which was either left unstimulated or stimulated with 100 ng/mL IL-6 (PeproTech EC, London, UK) for 15 min at 37°C. BD Phosflow Lyse/Fix and BD Phosflow Perm Buffer III (both BD Biosciences) were used as per the manufacturers’ instructions. IL-6R expression was assessed in whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturers’ instructions. Data were collected on a BD FACSCanto II (BD Biosciences) and analysed using FlowJo (Treestar, Ashland, Oregon, USA). The protocol followed for samples processed in Brisbane was similar, using anti-CD3-Pacific blue (UCHT1), and anti-Stat3 (pY705)-PE (4/P-STAT3; BD Biosciences), with a Gallios flow cytometer and Kaluza software for data acquisition/analysis (both Beckman Coulter). Flow-Set Pro Fluorospheres (Beckman Coulter) were used to normalise median fluorescence intensities (MFI) for pSTAT3 measurements between data sets.
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