Staphylococcal enterotoxin b

Manufactured by Toxin Technology

Sourced in United States

Staphylococcal enterotoxin B is a protein produced by the bacterium Staphylococcus aureus. It is a component of some laboratory equipment used for research and analysis purposes.

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Lab products found in correlation

Cd4 pe cy7, by BioLegend (2 mentions) Ifn γ apc, by BD (2 mentions) Tnf α fitc, by BD (2 mentions) Cd8 percp cy5, by BD (2 mentions) Cd69 pe texas red, by Beckman Coulter (2 mentions) Cd3 pacific blue, by BD (2 mentions) Brefeldin a, by Merck Group (2 mentions) Lsrii instrument, by BD (2 mentions) Human ab serum, by Gemini Bio (2 mentions) Recombinant soluble il 21 receptor, by R&D Systems (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Antibiotic cocktail, by Merck Group (2 mentions) Anti cd25 or isotype control mabs, by BioXCell (2 mentions) Streptolysin o, by Merck Group (1 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Fortessa, by BD (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Facsaria 3, by BD (1 mentions) Aim 5 media, by Thermo Fisher Scientific (1 mentions) Probenecid, by Thermo Fisher Scientific (1 mentions)

27 protocols using staphylococcal enterotoxin b

Flow Cytometry Assay for CD4+ and CD8+ T Cell Responses

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CD4⁺ and CD8⁺ T cell responses were measured from blood and tissues by flow cytometric ICS, as previously described.^{54 (link)} Briefly, 1 × 10⁶ mononuclear cells were incubated with Gag or Vif open-reading frame pools and the co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by addition of Brefeldin A (Sigma-Aldrich) for an additional 8 h. Co-stimulation without antigen served as a background control, while incubation with Staphylococcal Enterotoxin B (Toxin Technology) served as the positive control. The cells were then labeled with CD4 PE-Cy7 (Biolegend) and CD8 PerCP-Cy5.5 (BD Biosciences) and fixed with 2% paraformaldehyde. After permeabilization, the cells were stained with CD3 Pacific Blue, IFN-γ APC, TNF-α FITC (BD Biosciences, all), and CD69 PE-Texas Red (Beckman Coulter). The cells were fixed and flow cytometric analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star, Ashland, OR). In some cases, cells were CD25-depleted prior to setting up the ICS experiment to remove T regulatory cells (Miltenyi Biotec).

Hessell A.J., Jaworski J.P., Epson E., Matsuda K., Pandey S., Kahl C., Reed J., Sutton W.F., Hammond K.B., Cheever T.A., Barnette P.T., Legasse A.W., Planer S., Stanton J.J., Pegu A., Chen X., Wang K., Siess D., Burke D., Park B.S., Axthelm M.K., Lewis A., Hirsch V.M., Graham B.S., Mascola J.R., Sacha J.B, & Haigwood N.L. (2016). Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques. Nature medicine, 22(4), 362-368.

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Antigen-Specific Tfh Cell Responses

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Cryopreserved tonsillar cells were thawed and cultured in RPMI medium containing 10% Human AB serum (Gemini Bio-products, West Sacramento, CA). As a positive control, cells were stimulated with 100 ng/mL staphylococcal enterotoxin B (Toxin Technology, Sarasota, FL). For antigen-specific proliferation, cells were stimulated with Streptolysin O, which had been previously heat inactivated at 65°C for 20 minutes (Sigma, St. Louis, MO). Cells were labeled with Cell Trace Violet (Thermo Scientific, Waltham, MA) and cultured for 96 hours in medium supplemented with 4ng/mL IL-7 (Peprotech, Rocky Hill, NJ). Cells were labeled with fixable viability dye eFluor 780 (eBioscience). Tonsils were sorted using BD FACSAria III to isolate GC Tfh cells (Live CD19⁻, CD14⁻, CD16⁻, CD8a, CD4⁺CD45RA⁻CXCR5^hiPD-1^hi), mTfh cells (Live CD19⁻, CD14⁻, CD16⁻, CD8a, CD4⁺CD45RA⁻CXCR5⁺PD-1⁺), and non-Tfh cells (Live CD19⁻, CD14⁻, CD16⁻, CD8a, CD4⁺CD45RA⁻CXCR5⁻). Sorted cells were incubated at a 1 CD4⁺ T cell : 1 APC ratio with irradiated autologous lymphoblastoid cell lines created from each donor tonsil as APCs. Cells were cultured for 96 hours and acquired on BD Fortessa.

Dan J.M., Arlehamn C.S., Weiskopf D., Antunes R.D., Havenar-Daughton C., Reiss S., Brigger M., Bothwell M., Sette A, & Crotty S. (2016). A cytokine-independent approach to identify antigen-specific human germinal center Tfh cells and rare antigen-specific CD4+ T cells in blood. Journal of immunology (Baltimore, Md. : 1950), 197(3), 983-993.

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Sarcoidosis Blood Test Methodology

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EXAMPLE 1

Blood Test Methodology

Patients with biopsy-proven sarcoidosis and control subjects (including PPD+, BCG+ controls and subjects undergoing bronchoscopy) were recruited with informed consent and IRB approval. A whole blood stimulation INFγ-release assay was tested using full-length recombinant (rec)-mKatG and PPD as antigens. INFγ-release after 24 hrs was measured by ELISA in each condition and in a separate background control condition in which culture media was added. Staphylococcal enterotoxin B (Toxin Technology) was used as a positive control. Following pilot studies assessing optimal doses and conditions, a sarcoidosis diagnosis was determined by the following results: mKatG minus media (bkd)>100 pg/ml and mKatG>PPD.

US09977029B2. Diagnostic blood test for sarcoidosis (2018-05-22). None [US]. Inventors: David R. Moller [US].

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Treg Modulation of B-TFH Cell Response

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RPMI-1640 medium as described above was used for co-culture. FACS-purified 1
× 10⁵ human B cells and FACS-purified 5 ×
10⁴ human T_FH cells were seeded in round-bottom
96-well plates with or without FACS-purified 1 × 10⁵human CD25⁺Treg or LAG3⁺Treg in the presence of
2 μg ml⁻¹recombinant staphylococcal enterotoxin B (Toxin Technology). The culture
supernatants were harvested on day 12, and total IgG levels were analysed with
ELISA using Human IgG Quantitation Set kits (Bethyl Laboratories), according to
the manufacturer’s protocol.

Okamura T., Sumitomo S., Morita K., Iwasaki Y., Inoue M., Nakachi S., Komai T., Shoda H., Miyazaki J.I., Fujio K, & Yamamoto K. (2015). TGF-β3-expressing CD4+CD25−LAG3+ regulatory T cells control humoral immune responses. Nature Communications, 6, 6329.

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Tonsillar Cell Activation and PBMC Stimulation

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Cryopreserved tonsillar cells were thawed and cultured in serum free AIM-V media (Life Technologies, Grand Island, NY) overnight for 18 hours. Cells were stimulated with 10 μg/mL heat inactivated antibiotic-killed Group A Streptococcus, strain 5448. As a positive control, cells were stimulated with 1 μg/mL staphylococcal enterotoxin B (Toxin Technology, Sarasota, FL). Cells were cultured for 18 hours and acquired on BD Fortessa. PBMCs were thawed and cultured in complete RPMI 1640 with 5% human AB serum (Gemini Bioproducts) for 24 hours. Cells were stimulated with 2 μg/mL peptide pools or 10 μg/mL PHA.

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Tonsil Mononuclear Cell Analysis

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Human tonsils from patients undergoing routine tonsillectomy were obtained after informed consent and in accordance with the local ethics committee of the Charité University Medicine Berlin. Mononuclear cells were prepared by mechanical disruption of tissue and ficoll density centrifugation. The following monoclonal antibodies conjugated to Biotin, FITC, PE, Alexa Fluor 647, Alexa Fluor 700, or Pacific Blue were used for flow cytometry staining: OKT3 (anti-CD3), 91d6 (anti-CD4), 1H3 (anti-CD62L), FN50 (anti-CD69), KPL-1 (anti-PSGL-1), 3D12 (anti-CCR7), RF8B2 (anti-CXCR5), and F44 (anti-ICOS; Hutloff et al., 1999 (link)). For functional assays, tonsillar cells were cultured in round bottom tubes in RPMI 1640 in the presence of 20 pg/ml staphylococcal enterotoxin B (Toxin Technology) and either 20 µg/ml blocking anti–ICOS-L antibody HIL-131 (Khayyamian et al., 2002 (link)) or a murine IgG1 isotype control (18–1-16). To ensure immediate T/B contact, cells were centrifuged at 50× g for 5 min.

Weber J.P., Fuhrmann F., Feist R.K., Lahmann A., Al Baz M.S., Gentz L.J., Vu Van D., Mages H.W., Haftmann C., Riedel R., Grün J.R., Schuh W., Kroczek R.A., Radbruch A., Mashreghi M.F, & Hutloff A. (2015). ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2. The Journal of Experimental Medicine, 212(2), 217-233.

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Modulation of B-cell Help by T Cells

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Naive T cells from PBMCs (CD19⁻ CD4⁺ CD45RA⁺), memory T cells from BAL (CD19⁻ CD16⁻ CD14⁻ CD4⁺ CD45RA⁻), and non-Tfh memory cells (CD19⁻ CD4⁺ CD45RA⁻ CXCR5⁻) or Tfh cells (CD19⁻ CD4⁺ CD45RA⁻ CXCR5⁺) from tonsils were sorted on an ARIA II flow sorter. Sorted T cells were cocultured for 7 days with heterologous tonsillar memory B cells (CD19⁺ CD4⁻ IgD⁻ CD38⁻) at a 1:1 ratio in the presence of 4 ng/ml staphylococcal enterotoxin B (Toxin Technology) as described previously (18 (link)). To block T-cell help, an antibody against CD40L (clone TRAP1; 20 μg/ml) and/or recombinant soluble IL-21 receptor (R&D Systems; 10 μg/ml) were added.

Bauer L., Müller L.J., Volkers S.M., Heinrich F., Mashreghi M.F., Ruppert C., Sander L.E, & Hutloff A. (2021). Follicular Helper–like T Cells in the Lung Highlight a Novel Role of B Cells in Sarcoidosis. American Journal of Respiratory and Critical Care Medicine, 204(12), 1403-1417.

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Analysis of T-cell signaling cascades

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Anti-CD3 (clone OKT3), anti-ZAP-70 (clone 1E7.2), mouse IgG1 isotype control, and APC-conjugated anti-mouse IgG1 (clone M1-14D12) were from eBioscience. Rabbit anti-GFP (clone ab290) and goat anti-Rabbit IgG H&L (HRP) (clone ab6721) were from abcam. X-rhod-1-AM, probenecid, Dynabeads, and anti-β tublin antibody were from Invitrogen. Goat anti-mouse IgG peroxidase conjugated, and mouse anti-goat IgG H&L were from Thermo Scientifc. Rabbit anti-phosphoZAP-70 was from Cell Signaling. Mouse anti-phosphotyrosine 4G10 was from Millipore. Superantigen was a mix of recombinant staphylococcal enterotoxin E, staphylococcal enterotoxin A, staphylococcal enterotoxin B, and staphylococcal enterotoxin C3 coming from Toxin Technology. All chemicals and other reagents were from Sigma unless otherwise indicated.

Li K., Xiang X., Sun J., He H.T., Wu J., Wang Y, & Zhu C. (2016). Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-based Biosensor. Annals of biomedical engineering, 44(12), 3510-3521.

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Tonsillar Tfh and Peripheral Tph Cells Co-Culture

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Tonsillar follicular helper T cells (Tfh, CD19⁻CD4⁺CD45RA⁻CXCR5^high) from patients who underwent routine tonsillectomy or peripheral memory T helper cells from bronchoalveolar lavage (BAL) of sarcoidosis patients (mostly peripheral helper T cells, Tph, CD19⁻CD4⁺CD45RA⁻) were sorted on an ARIA II flow cytometry sorter (Becton Dickinson). Patient samples were obtained from the Unfallkrankenhaus Marzahn (tonsils) or the Charité Universitätsmedizin Berlin (BAL). Sorted T cells were co-cultured for 7 days with heterologous tonsillar memory B cells (CD19⁺CD4^−IgD⁻CD38⁻) at a 1:1 ratio in the presence of 4 ng/ml staphylococcal enterotoxin B (Toxin Technology) as described previously^{36 (link)}. To block T cell help, 20 μg/ml anti-CD40L antibody (clone TRAP1) and/or 10 μg/ml recombinant soluble IL-21 receptor (R&D Systems, Cat. 9249-R2) were added to the culture. Cells were acquired on an LSR II Fortessa flow cytometer (Becton Dickinson) and analysed using FlowJo version 10 software (Tree Star Inc.).
All antibodies utilised in the study are listed in Supplementary Table 3 for reference.

Ferreira-Gomes M., Chen Y., Durek P., Rincon-Arevalo H., Heinrich F., Bauer L., Szelinski F., Guerra G.M., Stefanski A.L., Niedobitek A., Wiedemann A., Bondareva M., Ritter J., Lehmann K., Hardt S., Hipfl C., Hein S., Hildt E., Matz M., Mei H.E., Cheng Q., Dang V.D., Witkowski M., Lino A.C., Kruglov A., Melchers F., Perka C., Schrezenmeier E.V., Hutloff A., Radbruch A., Dörner T, & Mashreghi M.F. (2024). Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow. Nature Communications, 15, 4182.

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Isolation and Activation of Murine T Cells

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CD4 and CD8 T cells were isolated from naïve balb/c mice using the MagniSort Mouse CD4⁺ or CD8⁺ T Cell Enrichment Kits (ThermoFisher Scientific) according to the manufacturer’s protocol. 5e4 purified T cells were co-cultured with 5e4 total A20 cells (a mixture of outlined genotypes) with 50 ng/mL staphylococcal enterotoxin B (Toxin Technology) for 48 hours before harvesting for analysis by flow cytometry.

Upadhyay R., Boiarsky J.A., Pantsulaia G., Svensson-Arvelund J., Lin M.J., Wroblewska A., Bhalla S., Scholler N., Bot A., Rossi J.M., Sadek N., Parekh S., Baccarini A., Merad M., Brown B.D, & Brody J.D. (2020). A critical role for fas-mediated off-target tumor killing in T cell immunotherapy. Cancer discovery, 11(3), 599-613.

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