EDTA-blood samples were stained with BD Multitest 6-color TBNK kit™ (lymphocyte subpopulations: B cells, CD8+cytotoxic T lymphocytes, CD4+T-helper lymphocytes, and NK cells) and analyzed by Facsverse™ flow cytometer. Blood samples were stained with BD Multitest 6-color TBNK kit™ (BD, Biosciences, USA). In brief, 20 μl of BD Multitest 6-color TBNK reagent was added into the bottom tube with 50 μl of EDTA anti-coagulated blood, gently mixed, and incubated for 15 min in the dark at room temperature (25 °C). Following incubation, 450 μl of 1xBD FACS lysing solution (BD, Biosciences, USA) was added, gently mixed, and incubated for 15 min in the dark at room temperature. The number of immune cells was determined using flow cytometry (BD Pharmingen, NJ, USA). The number of all T-lymphocyte subpopulations was normalized with total white blood cell count of each subject.
Multitest 6 color tbnk kit
Manufactured by BD
Sourced in United States
The BD Multitest 6-color TBNK kit is a laboratory diagnostic tool designed for the identification and enumeration of T cells, B cells, and natural killer cells in human whole blood samples. It utilizes flow cytometry technology to provide quantitative analysis of these key immune cell populations.
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Lab products found in correlation
Trucount tube, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Bn 2 automatic protein analyzer, by Siemens (1 mentions)
Facs lysing solution, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Immulite 1000 immunoassay analyzer, by Siemens (1 mentions)
Bricyte e6 flow cytometer, by Mindray (1 mentions)
Multitest 6 color tbnk reagent, by BD (1 mentions)
10 protocols using multitest 6 color tbnk kit
1
Comprehensive Lymphocyte Immunophenotyping
2
Lymphocyte Subpopulation Analysis
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Lymphocyte subpopulations, including CD19 positive B-cells, were determined in EDTA anticoagulated blood samples before treatment, and during all follow-up visits (3, 6, 10, 15, 20, 24, 30 and 36 months). Immunophenotyping of lymphocyte subpopulations was performed using the BD Multitest 6-color TBNK kit with BD Trucount Tubes for relative and absolute concentration determination (BD Biosciences). The samples were prepared according to the manufacturer’s instructions and immediately analyzed on a BD Canto II flowcytometer (BD Biosciences). Immunoglobulin levels in serum (IgG, IgA and IgM) were measured at all visits.
3
Comprehensive Biomarker Profiling Protocol
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Abbreviations are used to describe the biomarkers, and their reference values and categories are shown in Supplementary Table 1 .
MLR-Bf testing was performed with a Sysmex XN9000 and a Lambda Antigen Tray (ELISA). IgA, IgG, IgM, C3, C4, kapp, lamb, IgE, CRP, ASO, RF, ADNaseB, and SAA testings were performed on a SIEMENS BN II automatic protein analyzer using the original matching kit (Nephelometry). White blood cells, lymphocytes, B cells (CD3−CD19+), NK cells (CD3−CD16+CD56+), CD4+ T cells/lymphocytes (CD3+CD4+), CD8+T cells/lymphocytes (CD3+CD8+), CD3+CD4+/CD3+CD8+, and CIK cells (CD3+CD56+) were tested using the BD FACSCanto II and BD Multitest 6-color TBNK kit (flow cytometry). Anti-U1-nRNP, anti-Sm, anti-SSA-60kd, anti-Ro-52-52kd, anti-SSB, anti-Scl-70, anti-PM-Scl, anti-JO-1, anti-CENOP B, anti-PCNA, ANuA, AHA, anti-RIB-P, and AMA-M2 testings were performed using a AESKU HELIOS and AESKUSLIDES ANA Hep-2 kit (IIFA). Tests for ANA, anti-dsDNA IgG, and Anti_C1q were performed by a EUROBLOT Master and the original matching kit (Euroline).
MLR-Bf testing was performed with a Sysmex XN9000 and a Lambda Antigen Tray (ELISA). IgA, IgG, IgM, C3, C4, kapp, lamb, IgE, CRP, ASO, RF, ADNaseB, and SAA testings were performed on a SIEMENS BN II automatic protein analyzer using the original matching kit (Nephelometry). White blood cells, lymphocytes, B cells (CD3−CD19+), NK cells (CD3−CD16+CD56+), CD4+ T cells/lymphocytes (CD3+CD4+), CD8+T cells/lymphocytes (CD3+CD8+), CD3+CD4+/CD3+CD8+, and CIK cells (CD3+CD56+) were tested using the BD FACSCanto II and BD Multitest 6-color TBNK kit (flow cytometry). Anti-U1-nRNP, anti-Sm, anti-SSA-60kd, anti-Ro-52-52kd, anti-SSB, anti-Scl-70, anti-PM-Scl, anti-JO-1, anti-CENOP B, anti-PCNA, ANuA, AHA, anti-RIB-P, and AMA-M2 testings were performed using a AESKU HELIOS and AESKUSLIDES ANA Hep-2 kit (IIFA). Tests for ANA, anti-dsDNA IgG, and Anti_C1q were performed by a EUROBLOT Master and the original matching kit (Euroline).
4
Multiparametric Flow Cytometry Profiling of Lymphocyte Subsets
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Mature human lymphocyte subsets in peripheral whole blood were evaluated by multiparametric flow cytometry: T lymphocytes (CD3+), B lymphocytes (CD19+), Natural killer (NK) lymphocytes (CD3–CD16+ and/or CD56+), Helper/inducer T lymphocytes (CD3+CD4+) and Suppressor/cytotoxic T lymphocytes (CD3+CD8+).
The BD Multitest™ 6-Color TBNK kit which contains FITC-labeled CD3, clone SK7; PE-labeled CD16, clone B73.1 and PE-labeled CD56, clone NCAM16.2; PerCP-Cy 5.5–labeled CD45, clone 2D1 (HLe-1); PE-Cy7–labeled CD4, clone SK3; APC-labeled CD19, clone SJ25C1 and APC-Cy7–labeled CD8, clone SK1 was used with BD Trucount™ Tubes.
After sample incubation, a specific lyse/no wash procedure followed, and the cells were acquired using a BD FACSCanto™ II flow cytometer. Using cytometer-specific BD FACSCantoTM software (version 3.1), the results of the different cell subsets were obtained as a percentage of lymphocytes and the number of positive cells per microliter of blood (absolute count).
The BD Multitest™ 6-Color TBNK kit which contains FITC-labeled CD3, clone SK7; PE-labeled CD16, clone B73.1 and PE-labeled CD56, clone NCAM16.2; PerCP-Cy 5.5–labeled CD45, clone 2D1 (HLe-1); PE-Cy7–labeled CD4, clone SK3; APC-labeled CD19, clone SJ25C1 and APC-Cy7–labeled CD8, clone SK1 was used with BD Trucount™ Tubes.
After sample incubation, a specific lyse/no wash procedure followed, and the cells were acquired using a BD FACSCanto™ II flow cytometer. Using cytometer-specific BD FACSCantoTM software (version 3.1), the results of the different cell subsets were obtained as a percentage of lymphocytes and the number of positive cells per microliter of blood (absolute count).
5
Profiling Circulating Immune Cells
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Flow cytometry analyses were used to investigate the circulating immune cells via whole blood samples, which were taken before and following either abemaciclib, palbociclib, or ribociclib administration. The use of Becton Dickinson (BD) FACSCantoTM and BD FACSCanto IITM with BDTM Cytometer Setup and Tracking (CS&T) control enabled signals to be reproducible and comparable in spite of any fluctuation in environmental conditions. BDFACSC Diva Software was used for the acquisition and assessment of at least 1.5 × 106. The BD Multitest 6-color TBNK kit (BD) was used for the assessment of the following subpopulations: lymphocytes B, natural killer (NK), and T cells with CD4 and CD8 subpopulations.
6
Multicolor Flow Cytometry for Immune Cell Subset Quantification
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CD45+, CD19+, CD56+, CD3+, CD4+ and CD8+ T cells were counted using the BD Multitest 6-color TBNK kit (BD Biosciences Cat.-No. 337166, RRID: AB_2868707). 20 µl of the BD Multitest 6-color TBNK antibody were added into 5ml Trucount tubes and 50 µl whole blood (EDTA) was added. After an incubation step at 21°C for 15 min in the dark, 450 µL of pre-warmed (37°C) 1 x Red blood cell lysis buffer was added to the tubes and again incubated. Cell counts were acquired using a LSRFortessa X-20 and BD FACS DIVA software (v8.0) and data analysis was performed using FlowJo Software (v10.6).
7
Circulating Immune Cells Analysis
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A study of circulating immune cells was performed on samples coming from the OLTRE Study. The whole blood samples before and after olaparib treatment allowed to analyze circulating lymphocytes and their changes under therapy. Flow cytometry analysis was performed with dual or triple-laser flow cytometers Becton Dickinson (BD) FACSCanto™ and BD FACSCanto II™, with BD™ Cytometer Setup and Tracking (CS&T) control, in order to make the signals reproducible and comparable regardless of the variation in environmental conditions. Acquisition of at least 1.5 x 106 events was assessed by BDFACSC Diva software. The lymphocytes subpopulations (B, NK, T with CD4 and CD8 subpopulation) were assessed with BD Multitest 6-Color TBNK kit (Becton Dickinson™). More details are reported in Supplementary Methods
8
Lymphocyte Subpopulation Analysis
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Peripheral EDTA-anticoagulated blood samples were used to determine lymphocyte subpopulations (T, B and natural killer (NK) cells, the T cells further subdivided in CD4+ and CD8+ T cells) using the single platform method with BD Multitest 6-color TBNK kit (Becton Dickinson, California, USA) in BD TruCount tubes on FACSCanto II flow cytometers (Becton Dickinson, Belgium). Gating followed manufacturer’s instructions and data analysis was performed using BD FACSDiva software V.8.0.1.
9
Immune Subtyping of COVID-19 Patients
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Serum cytokines including interleukin (IL)-1, IL-2, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α were quantified by Immulite 1000 immunoassay analyzer (Siemens Healthcare). Absolute cell counts and relative percentage of CD3+ T cells, CD8+ cytotoxic T cells, CD4+ helper T cells, CD16+CD56+ NK cells, and CD19+ B cells of each patient were determined by BriCyte E6 flow cytometer (Mindray Medical) with Multitest 6-color TBNK kit (BD Biosciences). Counts of WBCs, neutrophils, lymphocytes, monocytes, eosinophils, and basophils (percentage) were extracted from the complete blood count assay by the XN-3000 blood analyzer (Sysmex). All these representative immune-related markers were hierarchically clustered by Pearson’s correlation distance, leading to two unsupervised immune-subtypes in the 188 COVID-19 patients (R package ConsensusClusterPlus version 1.56.0). The distinction of these two immune-subtypes was further illustrated by dimension reduction with t-distributed stochastic neighbor embedding (t-SNE) model (R package Rtsne version 0.15).
10
Multicolor Flow Cytometry Analysis
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