Cd28 cd49d

Manufactured by BD

Sourced in United States

CD28/CD49d is a lab equipment product that functions as a cellular activation reagent. It is used to stimulate T-cell activation and proliferation in cell culture applications.

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Lab products found in correlation

Cytofix cytoperm solution, by BD (6 mentions) Ifn γ and tnf α antibodies, by Miltenyi Biotec (4 mentions) Golgiplug, by BD (4 mentions) Cytoflex cytometer, by Beckman Coulter (4 mentions) Fast immune brefeldin a solution, by BD (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Cytofix cytoperm buffer, by BD (3 mentions) Facs lysing solution, by BD (3 mentions) 12 well tissue culture plate, by Corning (2 mentions) Brefeldin a golgiplug, by BD (2 mentions) Brefeldin a, by Merck Group (2 mentions) Cd56 bv650, by Miltenyi Biotec (2 mentions) Golgistop, by BD (2 mentions) Pepmix hcmva pp65, by JPT Peptide Technologies (1 mentions) Hsp70 peptide, by Abcam (1 mentions) Methanol, by Merck Group (1 mentions) Lsrfortessa x 20 flow cytometer, by BD (1 mentions) Intraprep permeabilization reagent, by Beckman Coulter (1 mentions)

15 protocols using cd28 cd49d

Intracellular Cytokine Profiling of Whole Blood

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Whole blood cell cultures were performed to determine the frequencies of intracellular cytokine-producing cells. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with SsAg, NIE, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d /CD28 (BD Biosciences) at 37°C for 6 or 18 h, for intracellular cytokine staining or ELISA respectively. Fast Immune Brefeldin A Solution (10μg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1x FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at -80°C until use. For cytokine neutralization experiments (n = 15), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5μg/ml) or anti-TGFβ (5μg/ml) or isotype control antibody (5μg/ml) (R& D Sytems) for 1 h following which NIE and brefeldin A was added and cultured for a further 23 h.

Anuradha R., Munisankar S., Bhootra Y., Jagannathan J., Dolla C., Kumaran P., Nutman T.B, & Babu S. (2016). IL-10- and TGFβ-mediated Th9 Responses in a Human Helminth Infection. PLoS Neglected Tropical Diseases, 10(1), e0004317.

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Intracellular Cytokine Profiling in Whole Blood

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Whole blood cell cultures were performed to determine the F_o of intracellular cytokine-producing cells.. Briefly, whole blood was diluted 1∶1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with BmA, Mf, PPD, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d/CD28 (BD Biosciences) at 37°C for 6 hrs. FastImmune Brefeldin A Solution (10 µg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1× FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at −80°C until use. The same procedure was used for both prospectively collected as well as for retrospectively stored samples. For cytokine neutralization experiments (n = 7), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5 µg/ml) or anti-TGFβ (5 µg/ml) or isotype control antibody (5 µg/ml) (R& D Sytems) for 1 h following which BmA and brefeldin A was added and cultured for a further 5 h.

Anuradha R., George P.J., Hanna L.E., Chandrasekaran V., Kumaran P.P., Nutman T.B, & Babu S. (2014). Parasite-Antigen Driven Expansion of IL-5− and IL-5+ Th2 Human Subpopulations in Lymphatic Filariasis and Their Differential Dependence on IL-10 and TGFβ. PLoS Neglected Tropical Diseases, 8(1), e2658.

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Cytokine Production in Whole Blood Cultures

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Whole blood cell cultures were performed to determine the F_o of intracellular cytokine-producing cells.. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with SsAg, NIE, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d /CD28 (BD Biosciences) at 37° C for 6 hrs. Fast Immune Brefeldin A Solution (10μg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1x FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at −80°C until use. For cytokine neutralization experiments (n=15), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5μg/ml) or anti-TGFβ (5μg/ml) or isotype control antibody (5μg/ml) (R & D Sytems) for 1 h following which NIE and brefeldin A was added and cultured for a further 23 h.

Anuradha R., Munisankar S., Dolla C., Kumaran P., Nutman T.B, & Babu S. (2015). Parasite antigen - specific regulation of Th1, Th2 and Th17 responses in Strongyloides stercoralis infection. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2241-2250.

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Intracellular Staining and Flow Cytometry

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Intracellular staining of pSAMHD1 was performed according to manufacturer’s instructions. Briefly, cells were fixed and permeabilized with methanol (Sigma Aldrich). After washing, cells were stained with anti-pSAMHD1-PE and then analyzed by flow cytometry. For intracellular staining of IFNγ, cells were treated for 4 h at 37°C with PepMix HCMVA pp65 (lower matrix protein 65) and IE1 (immediate-early-1 protein) (JPT Peptide Technologies, Berlin, Germany) to induce specific CD8 response against CMV or with Hsp70 peptide (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells, in the presence of brefeldin A and co-stimulator CD28/CD49D (BD Biosciences). Cells were then stained with antibodies against CD3 and CD8 conjugated to PE-Cy7 and APC-H7, respectively, or with CD3, CD56 and CD16 conjugated to APC, FITC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with an antibody against IFNγ-PE and then analyzed by flow cytometry.
Flow cytometry data acquisition was performed using BD LSR Fortessa X-20 flow cytometer (BD Biosciences) with BD FACSDiva software. Data analyses were carried out with FlowJo v10 software (TreeStar, Ashland, OR).

Vigón L., Rodríguez-Mora S., Luna A., Sandonís V., Mateos E., Bautista G., Steegmann J.L., Climent N., Plana M., Pérez-Romero P., de Ory F., Alcamí J., García-Gutierrez V., Planelles V., López-Huertas M.R, & Coiras M. (2020). Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection. Biochemical pharmacology, 182, 114203.

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Zika-specific T-cell Activation Assay

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1 × 10⁶ ZIKV-specific T-cell products were stimulated with ZIKV overlapping peptides (100 ng/ml), media alone, actin (negative control), or staphylococcal enterotoxin B (SEB, positive control) in the presence of brefeldin A and CD28/CD49d (BD Biosciences, San Jose, CA) for 6 hours. T-cells were fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and stained with IFN-γ and TNF-α antibodies (Miltenyi Biotec).

Hanajiri R., Sani G.M., Hanley P.J., Silveira C.G., Kallas E.G., Keller M.D, & Bollard C.M. (2019). Generation of Zika Virus-Specific T-Cells from Seropositive and Virus Naïve Donors for Potential Use as an Autologous or “Off the Shelf” Immunotherapeutic. Cytotherapy, 21(8), 840-855.

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PBMC Stimulation and Analysis

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PBMCs were isolated from heparinized peripheral blood following the protocol described before and seeded in a 96-well plate at a concentration of 2.5 × 10⁵ cells/well in RPMI-1640 medium, containing L-glutamine and sodium bicarbonate (Sigma-Aldrich, Saint Louise, MO, USA), and 10% FCS, stimulated with the three peptide pools S1, N (50 μL according to manufacturer’s instructions), and delta (2 μg/mL) for 18–20 h at 37 °C in a 5% CO₂ atmosphere. RPMI-1640 medium containing 10% FCS was used as a negative control for S1 and N pools, and an equimolar amount of 0.2% DMSO-as a negative control for the delta pool. Both the negative controls and test samples were cultured in the presence of co-stimulator CD28/CD49d (cat# 347690, BD, Biosciences, San Jose, CA, USA), as already described [15 (link)]. At the end of the overnight incubation, cells were harvested according to stimulation conditions and washed four times with PBS.

Aleksova M., Todorova Y., Emilova R., Baymakova M., Yancheva N., Andonova R., Zasheva A., Grifoni A., Weiskopf D., Sette A, & Nikolova M. (2023). Virus-Specific Stem Cell Memory CD8+ T Cells May Indicate a Long-Term Protection against Evolving SARS-CoV-2. Diagnostics, 13(7), 1280.

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Intracellular Cytokine Staining of T-Cells

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A total of 0.5 to 1 × 10⁶ VSTs were plated in a 96-well U-bottom plate and stimulated with pooled pepmixes or individual peptides (200 ng/peptide/well) or actin (irrelevant peptide control) in the presence of Brefeldin A (Golgiplug; BD Biosciences, San Jose, CA) and CD28/CD49d (BD Biosciences) for 6 h. T-cells were fixed, stained with fluorophore-conjugated antibodies against CD4, CD8 and TCRαβ, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and stained with IFN−γ and TNF-α antibodies (Miltenyi Biotec).

Harris K.M., Horn S.E., Grant M.L., Lang H., Sani G., Jensen-Wachspress M.A., Kankate V.V., Datar A., Lazarski C.A., Bollard C.M, & Keller M.D. (2020). T-Cell Therapeutics Targeting Human Parainfluenza Virus 3 Are Broadly Epitope Specific and Are Cross Reactive With Human Parainfluenza Virus 1. Frontiers in Immunology, 11, 575977.

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CTLA-4 Blockade Enhances TNF in PBMC

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PBMC were incubated in culture medium (RPMI 10%FCS), with 3 μg/ml CTLA-4 blocking antibody (BPS Bioscience, cat: 71212) or IgG1 isotype (Invitrogen, cat: 16-4714-85), at a concentration of 5x10⁵ cells/well, for 1hr at 37°C, 5% CO₂ in sterile polypropylene U-bottom microtiter plates control. Next, cells were stimulated for 24 hours with either 1 μg/mL Na-GST-1, or culture medium only, as negative control, in the presence co-stimulatory antibodies CD28/CD49d (BD Biosciences cat: 555725/555501), both at a final concentration of 2μg/ml. Supernatants were collected to measure tumor necrosis factor (TNF) by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences, cat: 555212), following manufacturer’s recommendation. It has to be noted that it was not possible to show an increase in cytokine production when intracellular cytokine staining was used.

Mouwenda Y.D., Betouke Ongwe M.E., Sonnet F., Stam K.A., Labuda L.A., De Vries S., Grobusch M.P., Zinsou F.J., Honkpehedji Y.J., Dejon Agobe J.C., Diemert D.J., van Leeuwen R., Bottazzi M.E., Hotez P.J., Kremsner P.G., Bethony J.M., Jochems S.P., Adegnika A.A., Massinga Loembe M, & Yazdanbakhsh M. (2021). Characterization of T cell responses to co-administered hookworm vaccine candidates Na-GST-1 and Na-APR-1 in healthy adults in Gabon. PLoS Neglected Tropical Diseases, 15(10), e0009732.

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Antigen-specific PBMC Stimulation Assay

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PBMCs were thawed and rested overnight prior to in vitro stimulation with vaccine candidate antigens. All samples met the minimum criteria of >80% viability (trypan blue staining) after thawing and resting.
PBMCs were cultured in 200μl RPMI 1640 (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS (Greiner Bio-One GmbH, Frickenhausen, Germany), 100 U/ml penicillin (Astellas, Tokyo, Japan), 10 μg/ml streptomycin, 1mM pyruvate and 2mM L-glutamine (all from Sigma-Aldrich, CA, USA). Five hundred thousand cells per well were stimulated in sterile polypropylene U-bottom microtiter plates with medium (negative control), 1ug/ml Na-GST-1, 1ug/ml Na-APR-1 (M74) or 200 ng/ml staphylococcus enterotoxin B (SEB) (positive control), all in the presence of co-stimulatory antibodies CD28/CD49d (BD Biosciences cat: 555725/555501) both at a concentration of 2μg/ml. Brefeldin A (10μg/ml, Sigma; cat: B7651) was added to the cells culture 5 hours post- stimulation and cells were cultured for a total of 24 hours at 37°C in the presence of 5% CO₂.

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Quantification of SARS-CoV-2-specific T cell Responses

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Testing of T cell responses was performed via stimulation of peripheral blood mononuclear cells with peptide libraries encompassing SARS-CoV-2 structural proteins as previously described [12 (link)]. Cells were then cultured for 10 days in 96-well plates with IL-4 (400 IU/mL) and IL-7 (10 ng/mL). On day 10, expanded viral specific T cells (VSTs) were harvested. 2 × 10⁵ VSTs were plated in a 96-well plate and re-stimulated with SARS-CoV-2 structural proteins pooled pepmixes or actin (negative control) with CD28/CD49d (BD Biosciences) and anti-CD107a- Pe-Cy7 antibody. After 1 h of stimulation, brefeldin A (Golgiplug; BD Biosciences, San Jose, CA, USA) and monensin (GolgiStop; BD Biosciences, San Jose, CA, USA) were added. Cells were then incubated for an additional 4 h. Cell viability was assessed using Live-Dead Aqua. Cells were surface stained with fluorophore-conjugated antibodies against CD3-BV785, CD4-BV605, CD8- BV421, TCRαβ-PerCP Cy5.5, TCRγδ- APC-Fire750, CCR7-FITC, CD45-RO-PE Dazzle, HLA-DR-Alexaflour700, and CD56-BV650 (Miltenyi Biotec; BioLegend). Cells were fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and subsequently stained with IFN-γ-APC and TNF-α-PE (Miltenyi Biotec). All samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA). The gating strategy for analysis is presented in Supplemental Fig. 2 .

Kinoshita H., Durkee-Shock J., Jensen-Wachspress M., Kankate V.V., Lang H., Lazarski C.A., Keswani A., Webber K.C., Montgomery-Recht K., Walkiewicz M., Notarangelo L.D., Burbelo P.D., Fuss I., Cohen J.I., Bollard C.M, & Keller M.D. (2021). Robust Antibody and T Cell Responses to SARS-CoV-2 in Patients with Antibody Deficiency. Journal of Clinical Immunology, 41(6), 1146-1153.

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