Clone b27

Manufactured by BD

Clone B27 is a serum-free and animal component-free supplement designed to support the growth and differentiation of neural and other cell types in cell culture. It is formulated to provide a defined and standardized set of essential nutrients, growth factors, and other components that are critical for maintaining the health and function of these cell types.

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Lab products found in correlation

Permeabilization buffer, by BioLegend (3 mentions) Clone mab11, by BD (3 mentions) Anti cd107a clone h4a3, by BD (3 mentions) Anti cd56 clone 5.1h11, by BD (3 mentions) Anti cd16 clone 3g8, by BD (3 mentions) Brefeldin a, by BioLegend (3 mentions) Golgistop, by BD (3 mentions) Lsrfortessa, by BD (3 mentions) Pta 6967, by American Type Culture Collection (2 mentions) Mip 1β, by BD (2 mentions) Fix perm, by Thermo Fisher Scientific (2 mentions) Golgistop, by Thermo Fisher Scientific (2 mentions) Anti cd107a antibody clone h4a3, by BD (2 mentions) Maxisorp 96 well plate, by Thermo Fisher Scientific (2 mentions) Brefeldin a, by Merck Group (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Interleukin 2 (il 2), by Roche (1 mentions) Tgf β, by Thermo Fisher Scientific (1 mentions) Golgiplug, by BD (1 mentions)

7 protocols using clone b27

NK Cell-Mediated ADNKA Assay for Antigen-Specific Responses

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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c ) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10⁴ cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.

Bates T.A., Lu P., Kang Y.J., Schoen D., Thornton M., McBride S.K., Park C., Kim D., Messer W.B., Curlin M.E., Tafesse F.G, & Lu L.L. (2022). BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age. medRxiv.

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ADNKA Assay for SARS-CoV-2 RBD Antibodies

Cited 1 time

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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c (link)) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10⁴ cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.

Bates T.A., Lu P., Kang Y.J., Schoen D., Thornton M., McBride S.K., Park C., Kim D., Messer W.B., Curlin M.E., Tafesse F.G, & Lu L.L. (2022). BNT162b2-induced neutralizing and non-neutralizing antibody functions against SARS-CoV-2 diminish with age. Cell Reports, 41(4), 111544.

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EBOV GPΔTM Antigen Binding Assay

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Recombinant EBOV GPΔTM antigen was coated onto a MaxiSorp 96-well plates (Nunc) at 300ng/well at 4°C overnight. Wells were washed with PBS and blocked with 5% BSA prior to addition of samples diluted 1:50 for 2h at 37°C. Unbound antibodies were removed by PBS wash, and NK cells enriched from the peripheral blood of human donors were added at 5×10⁴ cells/well in the presence of 4μg/ml brefeldin A (Sigma Aldrich), 5μg/ml GolgiStop (Life Technologies) and anti-CD107a antibody (Clone H4A3, BD Biosciences) for 5h. Cells were fixed and permeabilized with Fix/Perm (Life Technologies) according to manufacturer’s instructions to stain for intracellular IFNγ (Clone B27, BD Biosciences) and MIP-1β (Clone D21–1351, BD Biosciences). Cells were analyzed on a BD LSRII flow cytometer.

Gunn B.M., McNamara R.P., Wood L., Taylor S., Devadhasan A., Guo W., Das J., Nilsson A., Shurtleff A., Dubey S., Eichberg M., Suscovich T.J., Saphire E.O., Lauffenburger D., Coller B.A., Simon J.K, & Alter G. (2023). Antibodies against the Ebola virus soluble glycoprotein are associated with long-term vaccine-mediated protection of non-human primates. Cell reports, 42(4), 112402.

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Stimulation and Analysis of Tumor-Infiltrating Lymphocytes

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Tumor-infiltrating lymphocytes were stimulated for 5 h with plate-bound anti-CD3 (Miltenyi Biotec MACS GMP, clone OKT3) and soluble anti-CD28 (Miltenyi Biotec, clone 15E8) antibodies in the presence of GolgiPlug (BD Biosciences) in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. After stimulation, intracellular cytokine staining was performed to assess IFNy (BD Biosciences, clone B27) and TNFα (BD Biosciences, clone Mab11). For CD226 downregulation and induction assays, cells were stimulated for 6 days with TGF-β (50 ng/ml, Peprotech), IL15 (50 ng/ml, Miltenyi Biotec) or IL2 (20 UI/ml, Roche). For STAT inhibitor experiments, cells were incubated for 48 h with IL15 in the presence of STAT3i (CAS 501919-59-1, Sigma‒Aldrich) or STAT5i (CAS 2062-78-4, Sigma‒Aldrich).

Viot J., Abdeljaoued S., Vienot A., Seffar E., Spehner L., Bouard A., Asgarov K., Pallandre J.R., Renaude E., Klajer E., Molimard C., Monnien F., Bibeau F., Turco C., Heyd B., Peixoto P., Hervouet E., Loyon R., Doussot A., Borg C, & Kroemer M. (2023). CD8+ CD226high T cells in liver metastases dictate the prognosis of colorectal cancer patients treated with chemotherapy and radical surgery. Cellular and Molecular Immunology, 20(4), 365-378.

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Evaluating NK Cell Activation by SARS-CoV-2 RBD

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ADNKA was performed as described (26 (link), 133 (link)). ELISA plates were coated with recombinant RBD (300 ng/well) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serially diluted samples (1:10, 1:100, 1:1000) in duplicate for 2hours at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10⁴ cells/well) for 5hours with brefeldin A (Biolegend), Golgi Stop (BDBiosciences) and anti-CD107a (clone H4A3, BDBiosciences). Cells were stained with anti-CD56 (clone 5.1H11, BDBiosciences) and anti-CD16 (clone 3G8, BDBiosciences) and fixed with 4%PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BDBiosciences) and TNFα (clone Mab11, BDBiosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed. NK cell degranulation and activation were calculated as %CD56+CD107a+, IFNγ+ or TNFα+. Representative data from one dilution was chosen by the highest signal-to-noise ratio. Experiments were conducted two independent times.

Adhikari E.H., Lu P., Kang Y.J., McDonald A.R., Pruszynski J.E., Bates T.A., McBride S.K., Trank-Greene M., Tafesse F.G, & Lu L.L. (2023). Diverging maternal and infant cord antibody functions from SARS-CoV-2 infection and vaccination in pregnancy. bioRxiv.

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NK Cell Activation by MARV GP-specific Abs

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MARV GP-antigen was coated onto a MaxiSorp 96-well plate (Nunc) at 300 ng/well at 4°C overnight. Wells were washed with PBS and blocked with 5% bovine serum albumin (BSA) prior to addition of antibodies diluted to 10 μg/mL, or 5-fold serially diluted from 1 to 6.4 x 10⁻⁵ μg/mL, for 2 h at 37°C. Unbound antibodies were removed by PBS wash, and NK cells enriched from the peripheral blood of three different human donors were added at 5 x 10⁴ cells/well in the presence of 4 μg/mL brefeldin A (MilliporeSigma), 5 μg/mL GolgiStop (Life Technologies) and anti-CD107a antibody (Clone H4A3, BD Biosciences) for 5 h. Cells were fixed and permeabilized with Fix/Perm (Life Technologies) according to the manufacturer’s instructions to stain for intracellular IFNγ (Clone B27, BD Biosciences) and MIP-1β (Clone D21-1351, BD Biosciences). Cells were analyzed on an LSRII flow cytometer (BD Biosciences).

Ilinykh P.A., Huang K., Santos R.I., Gilchuk P., Gunn B.M., Karim M.M., Liang J., Fouch M.E., Davidson E., Parekh D.V., Kimble J.B., Pietzsch C.A., Meyer M., Kuzmina N.A., Zeitlin L., Saphire E.O., Alter G., Crowe JE J.r, & Bukreyev A. (2020). NON-NEUTRALIZING ANTIBODIES FROM A MARBURG INFECTION SURVIVOR MEDIATE PROTECTION BY FC-EFFECTOR FUNCTIONS AND ENHANCING EFFICACY OF OTHER ANTIBODIES. Cell host & microbe, 27(6), 976-991.e11.

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PBMC T cell allele presentation

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PBMC T cell cultures expanded with the dominant epitope for RV1196 or Rv0125 were tested for specific allele presentation, using RM3 cells transfected with MHC II alleles at a 1:1 or 2:1 ratio (i.e., T cell:transfected RM3 cell) (Fig. S1). Transfected RM3 cells were prepared for T cell testing via the addition of 1 μg/mL peptide to individual wells of a 96-well plate and were incubated for 90 min at 37°C and 5% CO₂. Following the incubation, the prepared T cells were added to each well of a 96-well plate and incubated for another 12 h in the presence of a Golgi plug inhibitor, namely, BFA (BD Biosciences, catalog number 555029). Stimulation plates were spun down and washed with 1× PBS, following 12 h of incubation. Flow cytometry staining was performed for viability (Live/Dead Fixable Blue Dead Cell Stain Kit, Thermo Fisher, catalog number L34962) and surface marker expression for CD3 (BD, clone SP34-2), CD4 (BD, clone L200), CD8 (BD, clone RPA-T8), HLA DR/DP/DQ (Bio-Rad clone Bu26; Beckman clone I3; BioLegend, clone Tü39), and the intracellular cytokines IFN-γ (BD, clone B27) and TNF (BD, clone Mab11), using standard flow cytometry and intracellular staining protocols. The samples were run on a BD LSRII or a Cytek Aurora (Cytek, Bethesda, MD, USA) and were analyzed using the FlowJo software package (BD, version 10).

Grant N.L., Kelly K., Maiello P., Abbott H., O’Connor S., Lin P.L., Scanga C.A, & Flynn J.L. (2023). Mycobacterium tuberculosis-Specific CD4 T Cells Expressing Transcription Factors T-Bet or RORγT Associate with Bacterial Control in Granulomas. mBio, 14(3), e00477-23.

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