Eomes

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Eomes is a laboratory equipment product by Thermo Fisher Scientific. It is a device designed for specialized applications in research and scientific analysis. The core function of the Eomes is to perform specific tasks as required by the user, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

10 protocols using eomes

Multiparametric Flow Cytometry Analysis

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The following antibodies were used for flow cytometry: CD8 (cat. no. 553035), B220 (553088), CD44 (553133), CD3ε (553066), and CD25 (102012) were purchased from BD; CD4 (cat. no. 100531), CD8 (100705), IFN-γ (505826), IL-2 (503808), T-bet (644808), Thy1.1 (202508), PD-1 (109109), CD45.1 (110722), and human NGFR (345108) were obtained from BioLegend; PD-1 (cat. no. 50–2073-82), FoxP3 (12-5773-82), TNF (17-7321-81), and Eomes (51-4875-82) were purchased from eBioscience.
Viability dye (eBioscience) or propidium iodide (PI; Sigma-Aldrich) were used in all experiments to exclude dead cells. For surface antigens, cells were stained with antibody cocktail in FACS buffer (PBS containing 0.5% BSA) on ice for 30 min. For intracellular cytokine staining, cells were stimulated with antigen or PMA (50 ng/ml) and Ionomycin (500 ng/ml; Sigma-Aldrich) for 3–5 h in the presence of GolgiStop (BD) before surface antigen staining. Next, cells were fixed and stained for cytokines using Cytofix/Cytoperm kit (BD) according to the manufacturer’s protocol. Staining of FoxP3, T-bet, and Eomes was performed using FoxP3/Transcription Factor Staining Buffer set (eBioscience) per the manufacturer’s instructions. Cells were processed using FACSCanto II (BD). All flow cytometry data were analyzed with FlowJo (Tree Star).

Hu Y., Kim J.H., He K., Wan Q., Kim J., Flach M., Kirchhausen T., Vortkamp A, & Winau F. (2016). Scramblase TMEM16F terminates T cell receptor signaling to restrict T cell exhaustion. The Journal of Experimental Medicine, 213(12), 2759-2772.

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RNA Isolation and qPCR Analysis

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Total RNA was isolated using TRIzol^® LS reagent (Invitrogen) and precipitated with isopropanol. All RNA samples were tested in duplicates, at 50 ng/well, using the One-Step Real-Time RT-PCR Master Mix Reagent (Applied Biosystems^®, Part Number 4309169). qPCR was performed and analyzed on the 7500 FAST Real-Time PCR System (Applied Biosystems^®). Slamf8 (Mm01293286_m1), Eomes (Mm01351985_m1), Zbtb16 (Mm01176868_m1), Irf4 (Mm00516431_m1), IL-4 (Mm00445259_m1), and the Eukaryotic 18S ribosomal RNA Endogenous Control TaqMan^® probes were purchased from Life Technologies (Carlsbad, CA, USA). The relative gene-specific fold change, normalized to 18S rRNA, was calculated using the 2^−ΔΔct method and expressed relative to WT levels (WT = 1).

De Calisto J., Wang N., Wang G., Yigit B., Engel P, & Terhorst C. (2014). SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors. Frontiers in Immunology, 5, 186.

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Quantitative RT-PCR Analysis of NK Cells

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Total RNA was isolated from the NK cells using the RNeasy Extraction Kit (Qiagen, Valencia, CA). RNA was reverse transcribed into cDNA using the Advantage RT-for-PCR Kit (Clontech, Mountain View, CA). Resultant cDNA (100 ng) was quantitated using TaqMan primers and probes as follows: CD226 (Hs00170832_m1), KLRK1 (Hs00183683_m1), NCR3 (Hs01553309_g1), GZMB (Hs00188051_m1), PRF1 (Hs00169473_m1), EOMES (Hs00172872_m1), JUN (Hs01103582_s1), SERPINE1 (Hs00167155_m1), SMAD7 (Hs00998193_m1), and GAPDH (Hs02786624_g1) (Life Technologies, Grand Island, NY). Each mRNA expression level was calculated as expression relative to GAPDH. Real-time PCR was performed on the 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, CA).

Fujii R., Jochems C., Tritsch S.R., Wong H.C., Schlom J, & Hodge J.W. (2018). An IL-15 superagonist/IL-15Rα fusion complex protects and rescues NK cell cytotoxic function from TGF-β1-mediated immunosuppression. Cancer immunology, immunotherapy : CII, 67(4), 675-689.

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Comprehensive Immune Cell Profiling

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Fluorochrome-conjugated antibodies against CD3, CD4, CD5, CD8a, CD27, CD45RA, CD45RO, CD122, Nur77, Eomes, NKG2A, NKG2D, KIR2D and KIR3DL1 were purchased from eBioscience, Biolegend or Miltenyi Biotec.

White J.T., Cross E.W., Burchill M.A., Danhorn T., McCarter M.D., Rosen H.R., O'Connor B, & Kedl R.M. (2016). Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner. Nature Communications, 7, 11291.

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Multi-Marker Immunophenotyping of T Cells

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Fluorochrome-labeled antibodies to CD8, CD44, PD-1, Lag3, CTLA4, 4-1BB, CD40, OX40, KLRG1, CD45.1, T-bet, and Eomes were purchased from eBioscience. Anti-4-1BB antibody (rat IgG2A) utilized for in vivo applications has been previously described¹⁸ (link) and was produced from hybridoma line 2A with permission from Dr. Lieping Chen (Yale University).

Song C., Sadashivaiah K., Furusawa A., Davila E., Tamada K, & Banerjee A. (2014). Eomesodermin is required for antitumor immunity mediated by 4-1BB-agonist immunotherapy. Oncoimmunology, 3, e27680.

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Comprehensive Immune Profiling by Flow Cytometry

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The flow cytometric was conducted as reported previously (27 (link)). For surface staining, the cells were cultured with antibodies specific for human cell surface markers CD3, CD4, CD8, CD103, CD69, LAG-3, PD-1, TIM-3, and CTLA-4 (564713, 564724,564116, 350225, 557745, 565775, 329920,
748820, 563931, all from BD Biosciences) for 30 min at 4°C. For intracellular cytokine staining (ICS), immune cells were first incubated in complete culture medium containing a leukocyte activation cocktail with GolgiPlug (BD Biosciences) at 37°C for 5 h. The cells were then stained with surface keratin and fixed with a BD Cytofix/Cytoperm kit for 20 min at 4°C. The cells were then stained with antibodies against IFN-γ, TNF-α (563416,559321, BD Biosciences), IL-2 and granzyme B (500310, 372221, Biolegend) for 30 min at 4°C. For nuclear factor staining, the cells were first stained with surface markers and then fixed with a transcription factor buffer set (BD Biosciences) for 50 min at 4°C. Next, the cells were stained with Runx3, T-bet (564814, 564141, BD Biosciences), TOX, and Eomes (50-6502-80, 25-4875-82, eBioscience) for 50 min at 4°C. All samples were detected using flow cytometry (BD LSR Fortessa, BD LSR Fortessa X20) and analyzed using FlowJo (v10.6.2, Tree Star, Ashland, USA).

Yang C., Qian Q., Zhao Y., Huang B., Chen R., Gong Q., Ji H., Wang C., Xia L., You Z., Zhang J, & Chen X. (2023). Fibrinogen-like protein 1 promotes liver-resident memory T-cell exhaustion in hepatocellular carcinoma. Frontiers in Immunology, 14, 1112672.

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Flow Cytometric Analysis of Murine Immune Cells

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Fluorescently conjugated antibodies directed toward mouse antigens CD45, CD3, CD11b, CD27, NK1.1, NKp46, and DX5 were purchased from BioLegend. Rat anti-mouse EOMES was purchased from eBioscience. Hamster anti-mouse CD49a was purchased from BD Biosciences. Surface antigens were stained in PBS containing 0.5% BSA and 1 mM EDTA. For intracellular staining of EOMES, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cell viability was assessed using 7-aminoactinomycin D (Tonbo Bioscience) in blood or Zombie-NIR Fixable Viability dye (BioLegend) in spleen and lung cell suspensions.

Rätsep M.T., Moore S.D., Jafri S., Mitchell M., Brady H.J., Mandelboim O., Southwood M., Morrell N.W., Colucci F, & Ormiston M.L. (2018). Spontaneous pulmonary hypertension in genetic mouse models of natural killer cell deficiency. American Journal of Physiology - Lung Cellular and Molecular Physiology, 315(6), L977-L990.

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Comprehensive Isolation and Characterization of Diverse Immune Cell Populations

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Single-cell suspensions were prepared from liver, spleen, bone marrow, uterus, salivary gland, thymus, and lung tissues. Liver lymphocytes were isolated at the interphase of a 40/60% Percoll gradient (1 (link)). Uterus and salivary gland were digested in the presence of DNase I and collagenase D or liberase TL (5 (link), 7 (link)), while lung digestion additionally incorporated trypsin inhibitor. Cells were stained with a LIVE/DEAD fixable dead cell stain kit (Invitrogen) and blocked with antibodies against CD16/CD32 (BD) prior to staining with antibodies for CD3, CD11b, CD27, CD43, CD122, human CD5, integrin α_v, KLRG1, Ly49G2, Ly49H, NKp46, and TRAIL (eBioscience); CD19, CD49a, CD49b, CD127, Ly49D, Ly-6G/Ly-6C (Gr-1), NK1.1, NKG2A/C/E, and TER-119 (BD). Cells were treated with cytofix/cytoperm kits prior to staining with antibodies for the transcription factors T-bet (eBioscience) and Eomes (eBioscience) or the cytokine TNF-α (BD).

Pikovskaya O., Chaix J., Rothman N.J., Collins A., Chen Y.H., Scipioni A.M., Vivier E, & Reiner S.L. (2016). Eomesodermin is Sufficient to Direct Type 1 Innate Lymphocyte Development into the Conventional Natural Killer Lineage. Journal of immunology (Baltimore, Md. : 1950), 196(4), 1449-1454.

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Multiparametric Phenotypic Analysis of T Cells

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The following antibodies were purchased from either BD or eBioscience: CD3e (145-2C11), CD4 (RM4-5), CD8α (53–6.7), TCRβ (H57-597), IL4, CD24 (M1/69), CD44 (IM7), CD122 (TM-β1), IFN-γ, Eomes (Dan11mag), and T-bet (4B10). APC-conjugated CD1d-PBS-57 (αGalCer analogue) loaded and unloaded tetramers were provided by the Tetramer Facility of the US National Institutes of Health. The monoclonal antibody against PLZF (D-9) was purchased from Santa Cruz Biotechnology, Inc. The Jmjd3 antibody was provided by Y. Shi (Boston Children’s Hospital, Boston, MA) and the Utx antibody (A302-374A) was purchased from Bethyl Laboratories. Anti-H3K4me3 (17–614) and anti-H3K27me3 (07–449) antibodies for ChIP-seq were purchased from Millipore.

Dobenecker M.W., Kim J.K., Marcello J., Fang T.C., Prinjha R., Bosselut R, & Tarakhovsky A. (2015). Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation. The Journal of Experimental Medicine, 212(3), 297-306.

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Multiparametric Immune Cell Profiling

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Cells were stained with fluorochrome-conjugated antibodies against the following antigens: CD56 (clone HCD56), CD3 (clone OKT3), CD57 (clone NK-1), CD62L (clone DREG-56), CD16 (clone 3G8), CD158a (HP-MA4), CD158b (DX27), NKB1 (DX9), Perforin (dG9), granzyme B (GB11), DNAM-1 (11A8), NKG2D (1D11), NKp80 (5D12), 2B4 (C1.7), TNF (MAb11), IFN-γ (clone B27) (all from Biolegend), NKG2C (134591), BLIMP1 (646702) (from R&D Systems), NKG2A (Z199) (from Beckman Coulter), ZEB2 (from Novus Biologicals), EOMES (WD1928), PLZF (Mags.21F7), SYK (4D10.1) (from eBiosciences), FcεR1γ (from Millipore), and T-BET (04-46) and NKp30 (p30-15) (from BD Pharmingen). All staining was done in combination with Live/Dead Fixable Dead Cell Stain (Thermo-Fisher). Detection of intracellular Perforin, granzyme B, T-BET, ZEB2, EOMES, BLIMP-1, PLZF, SYK, FcεR1γ, TNF and IFN-γ was performed following fixation and permeabilization (eBioscience) according to the manufacturer’s instructions. Cells were acquired on either an LSRII or Fortessa cytometer (BD Biosciences), and data was analyzed using FlowJo (TreeStar).

Cichocki F., Valamehr B., Bjordahl R., Zhang B., Rezner B., Rogers P., Gaidarova S., Moreno S., Tuininga K., Dougherty P., McCullar V., Howard P., Sarhan D., Taras E., Schlums H., Abbot S., Shoemaker D., Bryceson Y.T., Blazar B.R., Wolchko S., Cooley S, & Miller J.S. (2017). GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity. Cancer research, 77(20), 5664-5675.

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