Stimulation cocktail

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Stimulation cocktail is a specialized laboratory reagent designed to induce cellular activation and response. It contains a proprietary blend of compounds that stimulate specific signaling pathways within cells. The core function of this product is to facilitate the study of cellular dynamics and processes under controlled experimental conditions.

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Lab products found in correlation

Lymphoprep, by Axis-Shield (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Anti foxp3 pe cy7, by Thermo Fisher Scientific (1 mentions) Anti il 17 alexa647, by BioLegend (1 mentions) Anti cd4 alexa488, by BioLegend (1 mentions) Anti cd25 pe, by Thermo Fisher Scientific (1 mentions) Facsmelody, by BD (1 mentions) Ovalbumin (ova), by Merck Group (1 mentions) Golgistop monensin, by BD (1 mentions) Percoll density gradient, by GE Healthcare (1 mentions) Attune nxt cytometer, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Roche (1 mentions) Liberase, by Roche (1 mentions) Lsrii cytometer, by BD (1 mentions) Ab193189, by Abcam (1 mentions) Cyan adp, by Beckman Coulter (1 mentions) Dpbs 1, by Thermo Fisher Scientific (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Anti cd4 fitc, by Thermo Fisher Scientific (1 mentions)

11 protocols using stimulation cocktail

Comprehensive T-cell Phenotyping and Cytokine Analysis

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Surface phenotyping and intracellular cytokine analyses of T-cells were performed with freshly isolated PBMC. PBMCs were stained with antibodies for 30 min and analyzed immediately after washing with Dulbecco’s phosphate-buffered saline (DPBS 1×; Gibco, Life Technologies) in case of surface phenotyping. Isotype controls were used to confirm specificity of staining and to discriminate background staining.
To assess cytokine production of T-cells, PBMCs were stimulated with a stimulation cocktail (plus protein transport inhibitors; Ebioscience) for 4 h. stimulation cocktail consists of phorbol-12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin as protein transport inhibitors. Surface staining, fixation, and permeabilization (CytoFix/CytoPerm kit; BD Biosciences) followed. After fixation and permeabilization, PBMCs were stained intracellularly for IFNγ, IL-2, IL-10, IL-17A, and/or TNFα. Unstimulated PBMC served as controls. Stimulation induces a downregulation of CD4, thus CD4⁺ T-helper-cells (THs) were defined as CD3⁺CD8^neg T-cells.

Brinkhoff A., Sieberichs A., Engler H., Dolff S., Benson S., Korth J., Schedlowski M., Kribben A., Witzke O, & Wilde B. (2018). Pro-Inflammatory Th1 and Th17 Cells Are Suppressed During Human Experimental Endotoxemia Whereas Anti-Inflammatory IL-10 Producing T-Cells Are Unaffected. Frontiers in Immunology, 9, 1133.

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In Vitro T Cell Functional Assay

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In vitro T cell functional assay was performed on cryopreserved single-cell suspension. Thawed cell were washed and resuspended in pre-warmed RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin and GlutaMax (Gibco). Cells were stimulated in the presence of stimulation cocktail (PMA/ionomycin, brefeldin A, monensin) (eBioscience) for 5 hours. Then cells were harvested and subjected for flow cytometry analysis as described above.

Grabowski M.M., Watson D.C., Chung K., Lee J., Bayik D., Lauko A., Alban T., Melenhorst J.J., Chan T., Lathia J.D., Ahluwalia M.S, & Mohammadi A.M. (2023). Spatial immunosampling of MRI-defined glioblastoma regions reveals immunologic fingerprint of non-contrast enhancing, infiltrative tumor margins. medRxiv.

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Lymph Node Immune Cell Profiling

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Draining lymph nodes (LNs) were separated into single cells on postimmunization day 21 through a 70 µm cell strainer, and 5 × 10⁵ single cells, pretreated with stimulation Cocktail (plus protein transport inhibitors, Cat# 00-4975-93, eBioscience) for 6 hours, were dispensed into each tube along with intracellular/intranuclear staining fixation solution in 0.5% BSA. The antibodies staining was incubated with anti–IFN-gamma PE (Cat# 505807, BioLegend), anti–IL-17 Alexa647 (Cat# 506912, BioLegend), anti-CD4 Alexa488 (Cat# 100529, BioLegend), anti-CD25 PE (Cat# 12-0251-82, ThermoFisher), and anti-Foxp3 PE-Cy7 (Cat# 25-5773-80, eBioscience, San Diego, CA, USA). The stained cells were acquired on BD FACSMelody (BD Biosciences) and analyzed with FlowJo Software 10.5.3 (FlowJo LLC).

Lee J.Y., Kim S., Sohn H.J., Kim C.H., Kim T.G, & Lee H.S. (2023). Local Myeloid-Derived Suppressor Cells Impair Progression of Experimental Autoimmune Uveitis by Alleviating Oxidative Stress and Inflammation. Investigative Ophthalmology & Visual Science, 64(13), 39.

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Isolation of Cytokine-Producing B Cells

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We followed a previously described protocol to isolate cytokine-producing B cells (10 (link), 29 (link)). First, B cells were pre-enriched via depletion of non-B cells (Miltenyi Biotec) and cultured for 2 days under stimulation with 100 nM CpG oligonucleotides. Second, stimulation cocktail (eBioscience) was added to the cultures for 3 h to induce IL-10 secretion. Third, the viable cytokine-producing B cells were specifically isolated using a cytokine secretion assay according to the manufacturer’s instructions. Briefly, the pre-enriched B cells were incubated with IL-10 and TNFα catch reagents. The cells were subsequently labeled with IL-10 detection antibody or TNFα detection antibody conjugated to PE or APC, respectively. The IL-10- and TNFα-secreting cells were then sorted by FACS (BD Aria II). The purity of the cells was further confirmed by measuring the expression of IL-10 and TNFα by q-RT-PCR.

Zheng Y., Ge W., Ma Y., Xie G., Wang W., Han L., Bian B., Li L, & Shen L. (2017). miR-155 Regulates IL-10-Producing CD24hiCD27+ B Cells and Impairs Their Function in Patients with Crohn’s Disease. Frontiers in Immunology, 8, 914.

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CD4+ T Cell Activation Assay

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DC subsets were FACS-purified and pulsed with 2mg/ml ovalbumin (Grade VI, Sigma) for 2 hours before being washed extensively and co-cultured for 4 days with CFSE-labelled, naïve CD62L^hiCD25⁻CD4⁺ T cells FACS-purified from the lymph nodes of OTII mice. For intracellular cytokine staining, cultured cells were placed in a stimulation cocktail (eBioscience) containing PMA, ionomycin, brefeldin A and monensin for 4.5 hours, before fixation, permeabilisation and staining with antibodies against IL-17a, IFNγ and FoxP3 .

Scott C.L., Bain C.C., Wright P.B., Sichien D., Kotarsky K., Persson E.K., Luda K., Guilliams M., Lambrecht B.N., Agace W.W., Milling S.W, & Mowat A.M. (2014). CCR2+CD103− intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells. Mucosal Immunology, 8(2), 327-339.

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Profiling Tumor-Infiltrating Lymphocytes in Osteosarcoma

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Twenty-five freshly harvested osteosarcoma tumors (15 primary bone tumors and 10 PMs) were digested using an enzymatic cocktail (0.1% DNase I and Liberase 400 µg/mL; Roche). Leukocytes were enriched and isolated by Percoll density gradient (GE Healthcare). Cells were then stored in liquid nitrogen until further analysis. MFC was performed with either an LSRII cytometer (BD Biosciences) or an Attune NxT cytometer (Thermo Fisher Scientific). TILs were stimulated in the presence of stimulation cocktail (phorbol-12-myristate-13-acetate+ionomycin; eBioscience) and GolgiStop (Monensin; BD Biosciences) according to the manufacturer’s instructions. Samples were then stained for CD45, CD3, CD4, CD8, PD-1, TIM-3, and LAG-3. Intracellular staining for cytokines and transcriptions factors was then performed for Eomesodermin (EOMES), Foxp3, T-bet, and IFNγ. Not all samples were stained for the entire MFC panel (online supplemental table 1).

Ligon J.A., Choi W., Cojocaru G., Fu W., Hsiue E.H., Oke T.F., Siegel N., Fong M.H., Ladle B., Pratilas C.A., Morris C.D., Levin A., Rhee D.S., Meyer C.F., Tam A.J., Blosser R., Thompson E.D., Suru A., McConkey D., Housseau F., Anders R., Pardoll D.M, & Llosa N. (2021). Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes. Journal for Immunotherapy of Cancer, 9(5), e001772.

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Profiling Immune Cell Subsets by Flow Cytometry

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PBMCs were isolated using discontinuous Lymphoprep (Axis-ShieldPoCAs, Oslo, Norway) gradient centrifugation and were re-suspended in phosphate-buffered saline (PBS) containing 2% fetal bovine serum. Isolated PBMCs were collected by centrifugation, incubated with Fc-blocking antibody (purified anti-human CD16/32, BioLegend, San Diego, CA, USA) to prevent nonspecific binding and then incubated with the following antibodies (CD19: CF506236, BD Biosciences, CA, USA, 1:50; CD24: BDB563371, BD Biosciences, 1:50; CD27: BDB340424, BD Biosciences, 1:50; CD8: BDB564805, BD Biosciences, 1:50; IgA: ab193189, Abcam, CA, USA, 1:50) for 30 min on ice. After staining, cells were analyzed using CyAn-ADP (Beckman Coulter). Intracellular cytokine staining was performed by using the stimulation cocktail (eBioscience, San Diego, CA, USA) pre-stimulated for three hours as the manufacturer’s recommended protocol. Samples were analyzed by CyAn-ADP (Beckman Coulter) and data were processed using Flow Jo software (TreeStar, Ashland, OR).

Li W., Wang L., Shen C., Xu T., Chu Y, & Hu C. (2019). Radiation therapy-induced reactive oxygen species specifically eliminates CD19+IgA+ B cells in nasopharyngeal carcinoma. Cancer Management and Research, 11, 6299-6309.

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FACS Profiling of CD8+ T Cells from LLC Tumor Mice

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For FACS profiling of CD8⁺ T cells from LLC mice, primary tumors were harvested from mock or treated mice following one month of treatment. Tissue was then digested using a mixture of collagenase, hyaluronidase and DNase. The resulting single cell suspension was counted and plated in complete media (RPMI+10% FBS) with or without eBiosciences stimulation cocktail (1:1000) for 4 hours. Cells were then collected and blocked with Rat monoclonal anti-CD16/CD32 (Fc block Antibody) in PBS for 30 min at 4°C. Cells were then stained with antibodies against Live/dead, CD45, CD3, CD8a, and IFNγ for CD8⁺ T cells. Cells were then profiled by FACS.

Topper M.J., Vaz M., Chiappinelli K.B., DeStefano Shields C.E., Niknafs N., Yen R.W., Wenzel A., Hicks J., Ballew M., Stone M., Tran P.T., Zahnow C.A., Hellmann M.D., Anagnostou V., Strissel P.L., Strick R., Velculescu V.E, & Baylin S.B. (2017). Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer. Cell, 171(6), 1284-1300.e21.

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Lymphocyte Isolation and Th17 Characterization

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The lymphocytes in the thymus, spleen, and peripheral blood were extracted with lymphocyte separation medium and washed with PBS. The obtained mononuclear cells (1 × 10⁶/mL) were incubated with 2 μL of stimulation cocktail (eBioscience, San Diego, CA, USA) in RPMI 1640 with 10% fetal calf serum at 5% CO₂ and 37°C for 5 hours. After centrifuging and resuspending with PBS, the cells were stained with surface-specific Abs (anti-CD3e-APC and anti-CD4-FITC, all purchased from eBioscience (San Diego, CA, USA)) at 4°C in the dark for 30 min. After centrifuging and resuspending with PBS, the suspension was incubated with fixation/permeabilization solution (BD Biosciences, Sparks, MD, USA) and stained with anti-IL-17A-PE (eBioscience, San Diego, CA, USA) at 4°C in the dark for 30 min. Acquisition was done on a FACSCalibur flow cytometer (BD Biosciences, Sparks, MD, USA), and FlowJo software was used for analysis.

Zhang Y., Liang R., Xie A., Shi W., Huang H, & Zhong Y. (2020). Antagonistic Peptides That Specifically Bind to the First and Second Extracellular Loops of CCR5 and Anti-IL-23p19 Antibody Reduce Airway Inflammation by Suppressing the IL-23/Th17 Signaling Pathway. Mediators of Inflammation, 2020, 1719467.

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Cytokine Production in Immune Cell Cocultures

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Cocultures of 2.0 × 10⁵ B cells and 2.0 × 10⁵ CD4⁺ CD25⁻ T cells stimulated for 72 h with plate-bound CD3 mAb (0.5 µg/mL) were activated with stimulation cocktail plus protein transport inhibitors (eBioscience) for the last 5 h. In addition, 2.0 × 10⁵ B cells and 2 × 10⁵ CD14⁺ monocytes cocultured for 24 h were stimulated with LPS (100 µg/mL) for the final 5 h. The cells were stained for surface markers, permeabilized, stained intracellularly for IFNγ or TNFα and Foxp3 and analyzed by flow cytometry.

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