6 color tbnk reagent

Manufactured by BD

Sourced in Belgium

The 6-color TBNK Reagent is a laboratory tool designed for the identification and enumeration of T cells, B cells, and natural killer cells in biological samples. It provides a comprehensive analysis of the major lymphocyte subsets within a single test.

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4 protocols using 6 color tbnk reagent

SARS-CoV-2-Specific T Cell Assay

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PBMC were harvested from peripheral blood within 24 h of collection. PBMC were incubated with SARS-CoV-2 OPPs (ORF1ab-C, ORF3a, N, S, nsp12) at a concentration of 1 μg/mL and incubated at 37°C and 6.5% CO₂ for an hour. Later, the cells were washed and cultured in 48-well plates for 2 weeks supplemented with recombinant interleukin-2 (IL-2, Charles River Labs, USA) at 20 IU/mL every 2–3 days thereafter. On day 14, T cells were phenotyped and enumerated using the multitest 6-Color TBNK Reagent (BD Biosciences) and assessed for antigen-specificity using an intracellular cytokine assay. The remaining cells were cryopreserved in culture media with 10% dimethyl sulfoxide (Sigma-Aldrich) and transferred to liquid nitrogen.

Swaminathan S., Lineburg K.E., Ambalathingal G.R., Crooks P., Grant E.J., Mohan S.V., Raju J., Panikkar A., Le Texier L., Tong Z.W., Chew K.Y., Neller M.A., Short K.R., Gowda H., Gras S., Khanna R, & Smith C. (2022). Limited Recognition of Highly Conserved Regions of SARS-CoV-2. Microbiology Spectrum, 10(1), e02780-21.

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Lymphocyte Subset Analysis by Flow Cytometry

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Flow cytometry was used to measure the percentages of lymphocyte populations in whole blood using the monoclonal antibody conjugates (6 Color TBNK Reagent, CD3-PerCP-Cy5.5, CD4-APC, CD8-V450, CCR7-FITC, CD45RA-PE, CD19-PerCP-Cy5.5, CD27-PE, IgD-FITC, CD24-APC and CD38V450, all from BD Biosciences (Franklin Lakes NJ). The percentage of each patient’s lymphocyte subset was compared with normal control ranges for age that have either been established independently in the Boston Children’s flow cytometry laboratory. Lymphocyte proliferation assays were performed in three independent experiments.

Frugoni F., Dobbs K., Felgentreff K., Aldhekri H., Al Saud B.K., Arnaout R., Ali A.A., Abhyankar A., Alroqi F., Giliani S., Ojeda M.M., Tsitsikov E., Pai S.Y., Casanova J.L., Notarangelo L.D, & Manis J.P. (2015). A Novel Mutation in the POLE2 Gene Causing Combined Immunodeficiency. The Journal of allergy and clinical immunology, 137(2), 635-638.e1.

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Absolute Leukocyte Quantification via Trucount

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In order to determine the absolute leukocyte numbers, trucount staining was performed. Briefly, 50 µL EDTA blood and 20 µL of antibody mix (6-color TBNK Reagent and CD123 BUV395 from BD, CD15 PB, CD193 BV605 and HLA-DR BV785 from Biolegend, and CD14 PE-Cy5 from eBioscience) were added in BD trucount tubes and incubated for 15 min at room temperature in the dark. The samples were then fixed using 430 µL 1× BD FACS lysing solution (BD Biosciences).

Kvedaraite E., Hertwig L., Sinha I., Ponzetta A., Hed Myrberg I., Lourda M., Dzidic M., Akber M., Klingström J., Folkesson E., Muvva J.R., Chen P., Gredmark-Russ S., Brighenti S., Norrby-Teglund A., Eriksson L.I., Rooyackers O., Aleman S., Strålin K., Ljunggren H.G., Ginhoux F., Björkström N.K., Henter J.I, & Svensson M. (2021). Major alterations in the mononuclear phagocyte landscape associated with COVID-19 severity. Proceedings of the National Academy of Sciences of the United States of America, 118(6), e2018587118.

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Immune Cell Phenotyping after Anti-CD20 Incubation

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After a Ficoll separation, 1.8 × 10⁶ peripheral blood mononuclear cells (PBMC) from healthy donor were incubated overnight at 4°C with 20 μl of anti-CD20 monoclonal antibodies (rituximab, obinutuzumab, ocrelizumab, or ofatumumab) at 50 ng/ml; pre-incubated with 20 μl of serum from patients with anti-rituximab antibodies or from healthy donor diluted at 1:2. All samples were heated at 54°C for 30 min to inhibit complement activity. Cell viability was assessed using 10 μl of tryptan blue added to 90 μl of PBMC incubated in each condition. Numbers of dead and alive cells were counted in four different fields. The cells were washed three times in 3 ml of PBS (Cell Wash BD Biosciences^© Erembodegem, Belgium) at 4°C and incubated 30 min in darkness with a panel of antibodies specific for T, B, and NK cells: anti-CD3, anti-CD4, anti-CD8, anti-CD45, anti-CD19, anti-CD16 et CD56 (6-color TBNK Reagent BD Biosciences). Then, lysing Solution was added, and samples were incubated 10 min in darkness. The percentages of the T-lymphocytes (CD3+), B-lymphocytes (CD19+), and NK-cells (CD3- CD19-) were determined using Cytometer BD FACS Canto II.

Boyer-Suavet S., Andreani M., Lateb M., Savenkoff B., Brglez V., Benzaken S., Bernard G., Nachman P.H., Esnault V, & Seitz-Polski B. (2020). Neutralizing Anti-Rituximab Antibodies and Relapse in Membranous Nephropathy Treated With Rituximab. Frontiers in Immunology, 10, 3069.

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