Flow cytometric analyses were performed with 100 μl aliquots of EDTA-coagulated whole blood. Erythrocytes were lysed using ImmunoPrep Reagent System (Beckman Coulter), washed twice with PBS containing 2 % FBS, and then incubated for 15 min at room temperature with fluorochrome-conjugated surface antibodies including anti-HLA-DR-PerCp-Cy5.5 (clone L243), anti-CD16-PE-Cy7 (clone 3G8), anti-CD3-APC (clone SP34-2), CD8-APC (clone RPA-T8), anti-CD20-APC (clone 2H7), and anti-CD14-Pacific blue (clone M5E2). For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ buffer (BD Biosciences) for 30 min at room temperature. Cells were again washed and incubated with BD Cytoperm Plus™ buffer for 10 min on ice, then washed and incubated with DNase (30 mg) for 1 h at 37 °C, and washed and then stained for intracellular antigen with anti-BrdU-FITC (clone 3D4; BD Biosciences) and anti-Ki-67-PE (clone B56; BD Biosciences) for 20 min at room temperature. Samples were acquired on a BD FACS Aria (BD Biosciences) and analyzed with Tree Star Flow Jo version 9.6. Identification and quantitation of BrdU+ monocytes and CD14+ CD16+ monocytes was performed as previously described [16 (link)].
Anti ki 67 pe clone b56
Manufactured by BD
Sourced in United States
Anti-Ki-67-PE (clone B56) is a fluorescent-labeled antibody that binds to the Ki-67 protein, a proliferation marker. It can be used for the detection and analysis of proliferating cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytoperm plus buffer, by BD (1 mentions)
Anti cd20 apc clone 2h7, by Beckman Coulter (1 mentions)
Facsaria, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Immunoprep reagent system, by Beckman Coulter (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Facs lysing solution, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti hla dr apc clone l243, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Facsverse, by BD (1 mentions)
4 protocols using anti ki 67 pe clone b56
1
Multiparametric Flow Cytometry of Immune Cells
2
Multiparametric Flow Cytometry Assay
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Fresh whole blood was stained for 25 min at room temperature in the dark with the monoclonal antibodies anti-CD4-PerCp-Cy5.5 clone OKT4, anti-CD8-eFluor 450 clone OKT8, anti-HLA-DR-FITC clone LN3, anti-CD69-APC clone FN50, anti-CD38-PE-Cy7 clone HIT2, and fixable viability dye-eFluor 506 (Thermo Fisher Scientific, Wilmington, DE, United States). Erythrocytes were lysed (BD FACS Lysing Solution, BD Biosciences, San Jose, CA, United States) following the manufacturer’s instructions, and then the cells were permeabilizated and stained with anti- Ki-67-PE clone B56 (BD Biosciences, San Jose, CA, United States) and anti-CD3-Alexa eFluor 700 clone UCHT1 (Thermo Fisher Scientific, Wilmington, DE, United States) for 25 min at 4°C in the dark. The cells were fixed with 1% formaldehyde, acquired using a BD LSRFortessa™ flow cytometer, and, analyzed in FlowJo software (Becton–Dickinson, San Diego, CA, USA).
3
Cell Proliferation and Adhesion Assay
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The cell proliferation was probed with an antibody against the marker Ki67 (anti-Ki67 PE, clone B56, BD) with proper PE isotype control (clone B56, BD). Bone marrow MNCs and Jurkat cells were additionally tested to another proliferation marker, proliferating cell nuclear antigen (PCNA) with the PE mouse anti-human PCNA set (clone PC10, BD) with proper PE isotype control (PE mouse Ig2a, kappa, BD). HUVECs were probed for adhesion marker CD31 with PE mouse anti-human CD31 (clone WM59, BD) and PE isotype mouse IgG1 kappa control (clone MOPC-21, BD). All labeled cells were analyzed by flow cytometry with the FACS Canto II cytometer (BD) and Facs Diva software (BD).
4
Multiparameter Flow Cytometry of T Cells
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As described by Tian et al., fresh EDTA-K2 anticoagulated peripheral blood or frozen PBMCs were used for all the flow cytometry detection (Tian et al., 2015 (link)). The following anti-human flow cytometry antibodies cross-reactive with macaques were used: anti-CD3 APC—Cy7 (clone SP34–2), anti-CD8 PE-Cy7 (clone PRA-T8), anti-CD28 APC (clone CD28.8), anti-CD95 FITC (clone DX2) and anti-Ki67 PE (clone B56) were purchased from BD; anti-CD4 PerCP-Cy5.5 (clone OKT4), anti-CTLA-4 PE (clone BNI3), anti-HLA-DR APC (clone L243), and anti-PD-1 PE (clone EH12.2H7). They were purchased from Biolegend. Anti-CD38 FITC (Clone AT-1) was purchased from StemCell. CTLA-4 expression was detected by intracellular staining. Briefly, Cells were fixed and permeabilized with Cytofix/Cytoperm (BD BioSciences, USA) for 40 min at 4 °C in the dark after surface staining. Following two further washes with Perm/Wash buffer (BD BioSciences, USA), cells were intracellularly stained with anti-CTLA-4 for 1 h at 4 °C in the dark. Flow cytometric acquisitions were performed on a BD FACSVerse (BD BioSciences, USA) flow cytometer and all flow cytometric data were analyzed on FlowJoX10.0.7 software (FlowJo, USA).
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