Cells were stained with anti-human CD3-APC, anti-human CD4-PE, anti-human CD8-FITC, anti-human CD56-PE, anti-human CD279-APC, and anti-human CD137-PE (BD Biosciences, USA) for 15 mins at 4°C. They were then washed with and re-suspended in PBS. Events were acquired using a flow cytometer (Calibur, BD Bioscience) and analyzed by Cell Quest software (BD Biosciences).
Anti human cd4 pe
Manufactured by BD
Sourced in United States
Anti-human CD4-PE is a fluorescently-labeled antibody that binds to the CD4 protein expressed on the surface of certain immune cells. It is used for the identification and enumeration of CD4-positive cells in flow cytometry applications.
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Lab products found in correlation
Anti human cd8 fitc, by BD (2 mentions)
Anti human cd3 apc, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti human cd56 pe, by BD (1 mentions)
Calibur, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Anti human cd86 fitc, by BD (1 mentions)
Anti human cd3 percp, by BD (1 mentions)
Anti hla dr apc, by BD (1 mentions)
Anti human cd54 apc, by BD (1 mentions)
Rbc lysis buffer, by BD (1 mentions)
Anti human cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
Anti human il 10 bv421, by BioLegend (1 mentions)
Anti human cd8 pe cy7, by BD (1 mentions)
Anti human cd3 pb, by BioLegend (1 mentions)
Anti human cd14 pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti human ifnγ apc, by BD (1 mentions)
Apc mouse igg1, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti human cd4 pe
1
Multiparametric Flow Cytometry Analysis
2
Immunophenotyping of Immune Cells
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For cell surface and intracellular staining, standard protocols were followed using monoclonal antibodies. Cells and antibodies were co-incubated on ice in the dark for 30 minutes. For intracellular staining of cytokines, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) for 30 minutes on ice, followed by an additional 30-minute incubation with antibodies in the dark at 4°C. The following antibodies were used: Anti-HLA-DR-APC (BD, cat. no.: 339194), anti-human CD54-APC (BD, cat. no.: 561899), anti-human CD86-FITC (BD, cat. no.: 560958), anti-human CD3-Percp (BD, cat. no.: 552851), anti-Human CD4-PE (BD, cat. no.: 557344), anti-Human CD8-FITC (BD, cat. no.: 555366), anti-human Ki-67-APC (BioLegend, cat. no.: 350514), and anti-IFN-gamma-APC (BD, cat. no.: IC285A-100). Imaging flow cytometry was performed using the ImageStreamX MarkII quantitative imaging flow cytometer, while conventional flow cytometry was performed using the BD FACS Calibur flow cytometer.
3
Multicolor Flow Cytometry Immunophenotyping
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For surface marker staining, PBMCs were labeled with the following mAbs: anti-human CD14 PE-Cy7, anti-human CD33 PE, anti-human CD11b FITC, anti-human PD-L1 PerCp-eFluor (eBioscience), anti-human HLA-DR APC, anti-human CD8 PE-Cy7, anti-human CD4 PE (BD Biosciences), anti-human CD3 PB, anti-human CD15 BV421 (Biolegend). After incubation for 20 min at RT, the cells were analyzed using flow cytometer. For whole blood staining, 100 μl of fresh whole blood was labeled with above-mentioned antibodies for 20 min at RT, then lysed with red blood cell (RBC) lysis buffer (BD Biosciences), and subjected to flow cytometry.
For intracellular staining, the cells were fixed and permeabilized using Cytofix/Cytoperm Plus kit (BD Biosciences), and stained with the corresponding intracellular Ab, anti-human IFN-γ APC (BD Biosciences), anti-human IDO PerCp-eFluor (eBioscience), anti-human IL-10 BV421 and anti-human Arg1 PE (Biolegend). Data acquisition and analysis were performed by flow cytometer. Controls for each experiment included cells that were single stained for surface markers or intracellular proteins, unstained cells, and isotype-matched antibodies.
For intracellular staining, the cells were fixed and permeabilized using Cytofix/Cytoperm Plus kit (BD Biosciences), and stained with the corresponding intracellular Ab, anti-human IFN-γ APC (BD Biosciences), anti-human IDO PerCp-eFluor (eBioscience), anti-human IL-10 BV421 and anti-human Arg1 PE (Biolegend). Data acquisition and analysis were performed by flow cytometer. Controls for each experiment included cells that were single stained for surface markers or intracellular proteins, unstained cells, and isotype-matched antibodies.
4
Regulatory T Cell Expansion Assay
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Human PBMCs (ALLCELLS) were stained with anti-CD3 and Glycophorin A to enumerate T cells. Cells were cultured with rhIL-2 (100 IU ml−1) and anti-CD3/anti-CD28-coated Dynabeads (Life Technologies) at a ratio of 1 : 3 (cell : bead) in the presence or absence of 2.5 ng ml−1 rhTGF-β1 with or without either a-CTLA4-TGFβRII or a-CTLA-4 antibody (5 μg ml−1). Following culture for 24–48 h, cells were lysed and subjected to immunoblot analyses with the following primary antibodies: FOXP3, SMAD-2/3 (D7G7), or phospho-SMAD-2/3, and β-actin (Cell Signaling Technologies). On day 5, anti-CD3/anti-CD28 beads were magnetically removed and the number of Tregs (CD4+/CD25high/CD127low/FOXP3+) were enumerated by flow cytometry. Cells were stained extracellularly with anti-human CD4-PE, anti-human CD25-PE-CYTM5, and anti-human CD127-FITC antibodies (BD Biosciences). The cells were then permeabilized (BD Cytofix/Cytoperm Kit) and stained intracellularly with anti-human FOXP3-APC or the corresponding isotype control mouse IgG1-APC (eBioscience). The stained cells were washed twice with FACS buffer, run on the Gallios Flow Cytometer, and analyzed utilizing Kaluza Software (Beckman Coulter).
5
Antigen-Specific T Cell Proliferation Assay
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Antigen-specific T cell proliferation was determined in all patients by carboxy-fluorescein diacetate succinimidyl ester (CFSE) dilution assay as previously published [28 (link)]. After 5 days at 37°C, cells were washed and stained with anti-human CD4-PE (BD Biosciences Cat# 555347) and CD8-APC (BD Biosciences Cat# 555369) antibodies, and the viability dye 7-AAD. Flow cytometry analysis were performed on a FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA). The number of viable cells that had proliferated was determined by gating on the CD4+CFSElow and CD8+CFSElow. Proliferation index was calculated as the ratio between CD4+ or CD8+ proliferative frequency (%) in the presence of specific antigen and that in the absence of antigen, as previously reported [28 (link)].
6
Quantification of Apoptosis in T Cell Assays
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Peptide-pulsed T2 cells were incubated with effector cells in a 1:5 ratio overnight in SF-IMDM at 37°C and 10% CO2. For cell-surface staining, cells were washed twice and incubated with anti-human CD4-PE (BD Pharmingen, 1:100 dilution) for 30 min at RT, washed twice and fixed for intracellular staining using the Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm™) according to the manufacturer's instructions. Cells were washed twice with 1x BD Perm/Wash™ Buffer and intracellular staining was performed by incubating the cells with anti-active caspase-3 clone C92-605-Alexa Fluor→ 647 (BD Pharmingen, 1:200) for 40 min at 4°C. Finally, cells were washed twice with 1x BD Perm/Wash™ Buffer before resuspending in washing buffer for FACS-analysis (BD FACS Canto II). Data analysis was performed using FlowJo V10 software.
7
Macrophages Induce Treg Generation
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To assess whether macrophages in the presence of BM-MSCs generated Tregs, macrophages were first treated with BM-MSCs without cellular contact in a 0.4 µm Transwell system (Corning) for 6 days in the presence of M0, M1 or M2 inducer medium, after which they were cocultured with CD4 + T cells selected using CD4 MicroBeads (Miltenyi Biotec) in a 1:1 ratio (macrophages:T cells) in RPMI medium (HyClone) containing 10% FBS (Corning) in the presence of anti-CD2/CD3/CD28 beads (1:1 T cell ratio) (Miltenyi Biotec). After 5 days of coculture, T lymphocytes were harvested and blocked with FBS (Biowest) at 4 • C for 15 min. After blocking, anti-human CD4-PE (BD Biosciences) and anti-human CD25-FITC (BD Biosciences) antibodies were added. The cells were then permeabilized following the manufacturer's instructions with the FoxP3 staining buffer set kit (Invitrogen). After permeabilization, anti-human FoxP3-APC (Biolegend) was added. Acquisitions were made on a spectral flow cytometer Aurora (Cytek Biosciences) and analyzed with FlowJo V10 software (Supplementary Figure S1). CD4+ T lymphocytes cultured in the absence of macrophages were used as a control.
8
PBMC Staining and Flow Cytometry
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Human peripheral blood mononuclear cells (PBMCs) were plated at 1.105 cells per well in 96-well plates and then were labeled with GH-ALG (250 µg/mL) and/or with the following monoclonal antibodies used at a 1/100 dilution: anti-human TCRa/b-FITC (Clone T10B9.1A.31); anti-human CD2-PE (Clone RPA-2.10); anti-human CD3-PE (Clone HIT3a); anti-human CD4-PE (Clone RPA-T4); anti-human CD8-PE (RPA-T8); and anti-human CD28-PE (CD28.2) (all from BD Biosciences, Franklin Lakes, United States). Cell fluorescence was then measured by flow cytometry. A mean fluorescence intensity drop indicated competition between GH-ALG and the test antibody.
9
Enrichment and Transduction of CAR-T Cells
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The primary human T cells were enriched with the RosetteSep kit (Stem Cells Technology, Canada) by negative selection of unwanted cells from peripheral blood mononuclear cells (PBMCs) of healthy donors who provided signed consent. Subsequently, the cells were cultured and anti-CD3/anti-CD28 antibodies (ImmunoCult™ Human CD3/CD28 T Cell Activator, Stem Cells Technology, Canada) were added into the T cell culture medium (RPMI 1640, 10 % FBS, 1 % MEM NAA, 50 μM 2-mercaptoethanol, 300 IU/mL rhIL2) to activate primary T cells at day 0 for 24 h. Then, the T cells were transduced with GPC3-CAR lentivirus at a multiplicity of infection (MOI) of 10 with 4 μg/mL polybrene (Santa Cruz Biotechnology, USA). These genetically modified CAR-T cells were used for subsequent assays after proliferation. The expression of GPC3-CAR was analyzed by Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch, USA). The CD4+/ CD8+ T cell population of T cells and CAR-T cells were identified by flow cytometry using, APC anti-Human CD3, PE anti-Human CD4 and FITC anti-Human CD8 antibodies (BD, USA). The CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, USA) was also used to test the cell proliferation disparity between CAR-T cells and T cells.
10
Isolation and Characterization of PBMC
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Ficoll-Paque PLUS (GE, Boston, MA, USA, cat #17-1440-02), complete medium: TexMACS (Miltenyi Biotechnology, Bergisch Gladbach, Germany, cat #170-076-309) + IL-2 (Miltenyi Biotechnology, cat #130-097-748), PerCP5.5 anti-human CD3 (BD, Franklin Lakes, NJ, USA, cat #552852), PE anti-human CD4 (BD, cat #555347), APC anti-human CD8 (BD, cat #555369), APC anti-human EGFR (Biolegend, San Diego, CA, USA, cat #352906) FaDu (Minimum Essential Medium +10% fetal bovine serum [FBS]), HCT-RPMI 1640 (1640+10% FBS), FACS buffer: PBS +2.5% FBS, flow cytometry: Millipore Guava easyCyte HT (Merck Millipore, Burlington, MA, USA), CO2 incubator: ESCO MCO-20AIC (Ehimeken, Japan), cell counter: Cellometer Auto 1000 (Nexcelom Bioscience LLC, Lawrence, MA, USA) were all used.
Peripheral blood mononuclear cells were isolated via the Ficoll density gradient centrifugation method from whole blood samples of healthy volunteers at the Department of Blood Transfusion, Beijing Tongren Hospital. This study was approved by the ethics committee of Beijing Tongren Hospital (ethics number TRECKY2014-027), and the healthy volunteers whose blood samples were used had provided written informed consent. The FaDu cell lines and HCT-116 cell lines were purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, People’s Republic of China).
Peripheral blood mononuclear cells were isolated via the Ficoll density gradient centrifugation method from whole blood samples of healthy volunteers at the Department of Blood Transfusion, Beijing Tongren Hospital. This study was approved by the ethics committee of Beijing Tongren Hospital (ethics number TRECKY2014-027), and the healthy volunteers whose blood samples were used had provided written informed consent. The FaDu cell lines and HCT-116 cell lines were purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, People’s Republic of China).
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