Ebioscience cell stimulation cocktail plus protein transport inhibitor

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The EBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors is a laboratory reagent designed to activate and induce cytokine production in cell cultures. The product contains a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin, which stimulate cellular signaling pathways, as well as protein transport inhibitors to facilitate the detection of intracellular cytokines.

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Lab products found in correlation

Cd90.2 magnetic beads, by Miltenyi Biotec (3 mentions) Live dead fixable aqua dead cell staining kit, by Thermo Fisher Scientific (3 mentions) Cytofix cytoperm kit, by BD (3 mentions) Mouse lung dissociation kit, by Miltenyi Biotec (1 mentions) Anti fcγ rii 3, by BD (1 mentions) Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Brefeldin a, by BD (1 mentions) Histopaque 1119, by Merck Group (1 mentions) Cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions) Mouse liver dissociation kit, by Miltenyi Biotec (1 mentions) Facs lsrii cytometer, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Oligomycin, by Merck Group (1 mentions) Il 1β, by Merck Group (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) Facsaria 3 cell sorter, by BD (1 mentions)

Close spelling variants of this product

Cell Stimulation Cocktail (366 citations) Protein transport inhibitor (63 citations) Protein inhibitor cocktail (33 citations) EBioscience Cell Stimulation Cocktail (25 citations) Stimulation cocktail (11 citations) EBioscience Protein Transport Inhibitor Cocktail (8 citations) Protein inhibitor (8 citations) Cell Stimulation Cocktail (plus (3 citations) PLUSTM (3 citations) Cell Stimulation Cocktail (plus protein (2 citations)

10 protocols using ebioscience cell stimulation cocktail plus protein transport inhibitor

Isolation and Characterization of Lung Immune Cells

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To collect BALF immune cells, bronchoalveolar lavage (BAL) was performed as previously described.^{19 (link)} Whole-lung immune cells were isolated by enzymatic digestion of left lungs of cRaf-1-BxB (Raf-BxB) or C57Bl/6 WT mice by using the mouse lung dissociation kit and gentle
MACS dissociator (Miltenyi, Bergisch Gladmach, Germany) according to the manufacturer’s protocol.
After cell isolation via BALF or enzymatic digestion, erythrocytes were lysed and the cells were subsequently stimulated for 6 h with eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors) (Thermo Fisher Scientific, Schwerte, Germany). Afterward cells were stained for multi-color flow cytometry analysis (Gallios, Beckmann Coulter).
Prior to specific staining all samples were treated with anti-Fcγ RII/III (BD Pharmingen, Heidelberg, Germany) antibody. Following extracellular surface marker staining, samples were fixed and permeabilized for staining of intracellular proteins using the eBioscience™ Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific, Schwerte, Germany). The gating strategy to discriminate amongst the different immune cell subsets and their respective activation markers is exemplarily shown in supplementary Figures 2 and 3. Analysis of distinct immune cell populations was performed using the antibodies listed in supplementary Table 2.

Masemann D., Meissner R., Schied T., Lichty B.D., Rapp U.R., Wixler V, & Ludwig S. (2021). Synergistic anti-tumor efficacy of oncolytic influenza viruses and B7-H3 immune- checkpoint inhibitors against IC-resistant lung cancers. Oncoimmunology, 10(1), 1885778.

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Stimulation and Cytokine Analysis

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Tumor and TdLN cell suspensions were stimulated ex-vivo with 0.25 μM phorbol 12- myristate 13-acetate (PMA; Sigma) and 1 μg/mL Ionomycin (Sigma) at 37°C and 5% CO₂ for 3.5 hr in the presence of Brefeldin A (BD GolgiPlug), or alternatively, with eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific, 00-4975-93). After stimulation, cells were surface stained as mentioned above. Then, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation/permeabilization kit (BD) followed by intracellular staining of cytokines (IFN-γ, IL-2 and TNF-α) as indicated by the manufacturer protocol.

Flores-Santibañez F., Rennen S., Fernández D., De Nolf C., Van De Velde E., Gaete González S., Fuentes C., Moreno C., Figueroa D., Lladser Á., Iwawaki T., Bono M.R., Janssens S, & Osorio F. (2023). Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity. Frontiers in Immunology, 14, 1209588.

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Immune Cell Analysis of Pancreatic Tumors

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Samples were processed and analyzed as previously described (57 (link)). Pancreatic tumors were processed 24 hours after completion of the first cycle of therapy: 5 doses of TH-302 administered once daily and 2 doses of αVEGFR administered every 4 days. The pellet from the Histopaque-1119 (MilliporeSigma, 11191) separation was processed for endothelial cell analysis. CD8⁺ T cells were isolated using a magnetic-activated cell sorting CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec, 130-104-075), activated for 6 hours using the eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific, 00-4975-93), and analyzed by flow cytometry as previously described (56 (link)). Flow data were collected on a 5-laser, 18-color BD LSR II and analyzed using FlowJo version v10.7. A representative illustration of the immune cell gating scheme is provided in Supplemental Figure 5.

Liu A., Gammon S.T., Pisaneschi F., Boda A., Ager C.R., Piwnica-Worms D., Hong D.S, & Curran M.A. (2024). Hypoxia-activated prodrug and antiangiogenic therapies cooperatively treat pancreatic cancer but elicit immunosuppressive G-MDSC infiltration. JCI Insight, 9(1), e169150.

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Multimodal Immune Profiling in Mice

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On days 10 and 21, cohorts of mice were euthanized and splenocytes, whole liver, and lamina propria of the colon were harvested for flow cytometric analysis. Hepatic tissue and colonic lamina propria were digested into a single cell suspension using a commercial mouse liver dissociation kit (Miltenyi Biotec) and mouse lamina propria tissue dissociation kit (Miltenyi Biotec). To select only the donor T cells, a specific gating strategy was used (Sup. Fig. 1). A complete list of antibodies used is listed in Supplemental Table 1. For cytokine evaluation, splenocytes or cells from colonic lamina propria were incubated for 5 hours with eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) (Thermo Fisher Scientific) for T cell stimulation and protein transport inhibition. Cells were then stained with surface antibodies, permeabilized, fixed, stained with intracellular antibodies and analyzed within 24 hours. Analysis was performed with a FACS LSRII cytometer; FACSDiva software (BD Pharmingen) data analysis was performed using FlowJo (Tree Star).

Zitzer N.C., Snyder K., Meng X., Taylor P.A., Efebera Y.A., Devine S.M., Blazar B.R., Garzon R, & Ranganathan P. (2018). MicroRNA-155 modulates acute graft-versus-host disease by impacting T cell expansion, migration, and effector function. Journal of immunology (Baltimore, Md. : 1950), 200(12), 4170-4179.

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Isolation and Activation of T Cells from Murine Models

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T cells from the spleen of OT-I and OT-II mice or lung tumors of treated KP mice were isolated from single-cell suspensions using CD90.2 Magnetic beads (Miltenyi) according to the manufacturer’s protocol. For ex-vivo T cell activation, 1 × 10⁶ T cells isolated from lung tumors were stimulated at 37°C with eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (ThermoFisher Scientific) for 6 hours. Cells were washed and stained for intracellular cytokines using BD cytofix/cytoperm kit (BD Biosciences) according to the manufacturer’s instructions at the end of the 6-hour culture. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against IL-2, TNF-α, CD107a, and IFN-γ, or isotype-matched antibodies after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, CA).

Bag A., Schultz A., Bhimani S., Stringfield O., Dominguez W., Mo Q., Cen L, & Adeegbe D. (2022). Coupling the immunomodulatory properties of the HDAC6 inhibitor ACY241 with Oxaliplatin promotes robust anti-tumor response in non-small cell lung cancer. Oncoimmunology, 11(1), 2042065.

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Metabolic Regulation of Cytokine Production

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5 x 10⁶ SiLP cells were incubated in 250 μl of complete media containing 20 ng/ml IL-1β and IL-23 (R&D Systems) for 2 hours at 37°C, 5% CO₂. After 2 hours, cells received 50 ul of complete media containing eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (ThermoFisher) (2 μl/ml) on top and incubated for a further 3 hours. To measure the impact of metabolic inhibition on cytokine production, 2-DG (2.5 mM; Merck) or Oligomycin (1 μM; Merck) was added when adding IL-1β/-23. After incubation, cells were centrifuged, and cells were used for antibody staining for flow cytometry.

King J.I., Melo-Gonzalez F., Malengier-Devlies B., Tachó-Piñot R., Magalhaes M.S., Hodge S.H., Romero Ros X., Gentek R, & Hepworth M.R. (2023). Bcl-2 supports survival and metabolic fitness of quiescent tissue-resident ILC3. Mucosal Immunology, 16(5), 658-670.

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Immune Profiling of Tumor Microenvironment

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The tumors, spleens, or draining lymph nodes were sheared, while tumors were further digested by a Tumor Dissociation Kit (Miltenyi, Cat No.130-096-730). Then, the cell mixture was passed through 70-μm nylon mesh cell strainers (WHB-70 μm) to obtain a single-cell suspension. Erythrocytes were extracted with cell lysis buffer (BD Biosciences, Cat. No. 20180816). To perform intracellular cytokine staining, the obtained cells were activated with eBioscience™ Cell Stimulation Cocktail Plus Protein Transport Inhibitors (Invitrogen, Cat. No. 00-4975-93) for 6 h. The single-cell suspension was stained with viability dye (Zombie violet fixed active kit, Biogene, Cat. No. 423114) to differentiate dead cells from living cells. Afterward, the percentages of various immune cells in the tumor or spleen were identified using a FACS flow cytometer (BD FACSAria™ III Cell Sorter, BD Biosciences, San Jose, CA, USA) and examined by FlowJo software.

Yu L., Xie H., Wang L., Cheng M., Liu J., Xu J., Wei Z., Ye X., Xie Q, & Liang J. (2023). Microwave ablation induces abscopal effect via enhanced systemic antitumor immunity in colorectal cancer. Frontiers in Oncology, 13, 1174713.

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Intracellular Cytokine Profiling of Tumor-Infiltrating T Cells

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T cells from lung tumors of treated mice were isolated from single cell suspensions using CD90.2 Magnetic beads (Miltenyi) according to the manufacturer's protocol. 1 x 10 6 isolated T cells were then stimulated at 37°C with eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (Invitrogen) for 6 hours. Cells were washed and stained for intracellular cytokines using BD cytofix/cytoperm kit (BD Pharmingen) according to the manufacturer's instructions at the end of the 6-hour culture. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against IL-2, TNFα, CD107a, and IFN-γ, or isotype-matched antibodies after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, CA).

Bag A.K., Schultz A., Bhimani S., Dominguez W., Cen L., & Adeegbe D.O. (2021). The immunomodulatory properties of the HDAC6 inhibitor ACY241 supports robust anti-tumor response in NSCLC when coupled with the chemotherapy drug Oxaliplatin.

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Intracellular Cytokine Staining of Activated T Cells

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Purification of T cells or T cell subsets from spleen or resected tumors was performed using CD90.2 magnetic beads (Miltenyi) according to the manufacturer’s protocol or via cell sorting using FACSAria (BD Biosciences). For ex vivo T cell activation, 1×10⁶ T cells were stimulated at 37°C with eBioscience Cell Stimulation Cocktail plus protein transport inhibitor (Thermo Fisher Scientific) for 6 hours. Cells were washed and stained for intracellular cytokines using BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against interferon (IFN)-γ, granzyme B, and tumor necrosis factor (TNF)-α after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, California, USA).

Noyes D., Bag A., Oseni S., Semidey-Hurtado J., Cen L., Sarnaik A.A., Sondak V.K, & Adeegbe D. (2022). Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells. Journal for Immunotherapy of Cancer, 10(5), e004605.

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Assessing Antigen-Specific Immune Responses

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In order to assess antigen-specific responses, PBMC were first labeled using the CellTrace® violet proliferation kit (Invitrogen, Thermo Fisher), according to manufacturer's recommendations. Following labeling, 1 × 10⁶ cells were plated onto 96-well, flat bottom plates and left unstimulated, or stimulated with PPDb (5 μg/well), or Concanavalin A (ConA, 0.5μg/well, Sigma-Aldrich), in a total volume of 200 μl. Cells were then incubated at 37°C with 5% CO₂ for 7 days. In order to assess intracellular cytokine production, cultured PBMC were treated with either a 1 × solution of eBioscience™ Protein transport inhibitor (500 × Brefeldin A, Thermo Fisher) or with a 1 × solution of eBioscience™ Cell Stimulation cocktail plus Protein transport inhibitor [500 × phorbol 12-myristate 13-acetate (PMA), ionomycin, Brefeldin A] (Thermo Fisher) and incubated overnight for ~16 h prior to harvest on day 7 (24 (link)).

Boggiatto P.M., Kanipe C.R, & Palmer M.V. (2021). Enhanced Detection of Mycobacterium bovis-Specific T Cells in Experimentally-Infected Cattle. Frontiers in Veterinary Science, 8, 676710.

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