Ef780

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EF780 is a laboratory centrifuge that provides high-speed separation of biological samples. It has a maximum rotor speed of 14,000 rpm and a maximum relative centrifugal force (RCF) of 30,130 x g. The EF780 is designed for use in a variety of applications, including cell separation, nucleic acid extraction, and protein purification.

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Lab products found in correlation

Lsrfortessa, by BD (4 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions) Anti il 17a pe, by Thermo Fisher Scientific (2 mentions) Golgistop, by BD (2 mentions) Ef506, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Avantor (2 mentions) Na2edta, by Avantor (2 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Lonza (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Anti cd3 clone okt3, by BioXCell (1 mentions) Anti cd28 clone 9, by BioXCell (1 mentions) Hepes buffer, by Lonza (1 mentions) Anti ifn γ percp cy5, by Thermo Fisher Scientific (1 mentions) Anti il 4 fitc, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Lonza (1 mentions) Human ab serum, by Merck Group (1 mentions)

Spelling variants of this product

Fixable Viability Dye eF780 (19 citations) CD27 APC-ef780 (3 citations) LiveDead-eF780 (3 citations) Anti-CD4 APC-ef780 (2 citations) FVD) ef780 (2 citations) APC-eF780-coupled M1/70 (antiCD11b) (2 citations) Anti-F4/80-eF780 (2 citations) Anti-CD8-eF780 (2 citations) EF780 viability dye (2 citations) CD29-APC-eF780 (2 citations)

17 protocols using ef780

Differentiation and Cytokine Profiling of Human CD4+ T Cells

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Naive and memory human CD4⁺ T cells were cultured on anti-CD3 (clone OKT3, 10 μg/mL; BioXCell) and anti-CD28 (clone 9.3, 2 μg/mL; BioXCell) coated plates with 20 U/mL IL-2 in RPMI 1640 (Lonza), supplemented with 10% heat-inactivated FCS (BioSera), penicillin/streptomycin and 2 mM L-glutamine (both Lonza), 2-mercapto-ethanol (Gibco), HEPES buffer (Lonza), 100 mM sodium pyruvate (Gibco), and and 1% nonessential amino acids (Sigma). Cells were split and media replenished as necessary.
Th1, Th2, and Th17 cells were cultured with autologous irradiated APCs (CD4⁻ cells) at a 1:5 ratio, with 100 U/mL IL-2 and 1 μg/mL anti-CD3 (OKT3) in X-VIVO 15 media (Lonza), supplemented with 5% human AB serum (Sigma), penicillin/streptomycin, and L-glutamine. GSK-3 inhibitors were added as above and cells were split as necessary. To determine cytokine production, on day 7 of T-cell culture, cells were stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 4 h, in the presence of Golgi Stop. Cells were stained with fixable viability dye ef780 (eBioscience) and surface-stained with anti-CD4 Alexa700. Following fixation and permeabilization, intracellular staining for anti-IL-10 ef660, anti-IFNγ PerCPCy5.5, anti-IL-17A PE, and anti-IL-4 FITC (all eBioscience) was carried out.

Hill E.V., Ng T.H., Burton B.R., Oakley C.M., Malik K, & Wraith D.C. (2015). Glycogen synthase kinase-3 controls IL-10 expression in CD4+ effector T-cell subsets through epigenetic modification of the IL-10 promoter. European Journal of Immunology, 45(4), 1103-1115.

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Phenotyping Human Peripheral Blood Mononuclear Cells

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Fresh PBMCs from lithium-heparinised blood were isolated using Ficoll density gradient centrifugation. After collection, PBMCs were frozen and stored in liquid nitrogen until further use. PBMCs were thawed according to protocol and then stained with fixable viability dye eF780 (eBioscience) for 10 min at 4 °C. Then, the cells were stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8, CCR9, CXCR5, PD-1, ICOS, CD27 and CD45RO for 25 min at 4 °C (details of the antibodies used can be found in Supplementary Table S1). Fluorescence minus one (FMO) controls were taken along to check marker expression. All samples were acquired on a BD LSRFortessa (BD Biosciences) using BD FACSDiva software v.8.0.1. (BD Biosciences). FlowJoTM v10.8 Software (BD Life Sciences) was used for data analysis.

Hinrichs A.C., Kruize A.A., Lafeber F.P., Leavis H.L, & van Roon J.A. (2023). CCR9/CXCR5 Co-Expressing CD4 T Cells Are Increased in Primary Sjögren’s Syndrome and Are Enriched in PD-1/ICOS-Expressing Effector T Cells. International Journal of Molecular Sciences, 24(15), 11952.

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Multiparametric Flow Cytometry Analysis of Immune Cell Signatures

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Cells were stimulated with medium containing PMA (5ng/mL), ionomycin (500 ng/mL), and GolgiStop (BD Biosciences) for 3 h at 37°C. Cells were stained with fixable viability dye ef780 (eBioscience) and then surface-stained with anti-CD4 Alexa700. Following incubation with IC fixation buffer (eBioscience), cells were stained intracellularly with anti-IFN-γ PECy7, anti-IL-10 allophycocyanin, anti-IL-17A PE, and/ or anti-IL-4 PE (all eBioscience). For intracellular transcription factor staining, cells were fixed by incubation with the FoxP3 Staining Buffer Set (eBioscience). The antibodies anti-CD4 FITC, anti-Nfil3 PE, and anti-c-Maf ef660 and their corresponding isotype controls (all eBioscience) were used. Fluorescence intensity was measured on a BD LSR II flow cytometer and analyzed using FlowJo (Tree Star Inc.) software. FACS gating strategy is shown in Supporting Information Method 1.

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Comprehensive Immune Cell Profiling

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Single cell suspensions were blocked with Fc-Block (BioXCell) in FACS buffer and surface stained with the following monoclonal antibodies: CD4 (Biolegend, RM4-5), CD8 (eBioscience, 53-6.7), CD19 (6D5, Biolegend), CD25 (BD, PC61), CD44 (eBioscience, IM7), CD62L (MEL-14, eBioscience), CD90.2 (eBioscience, 53–2.1), γδTCR (eBioscience, eBioGL3), Gr-1 (BD, RB6-8C5), F4/80 (eBioscience, BM8). To exclude dead cells, cells were also stained with the fixable viability dye ef780 (eBioscience).
For T_reg stainings, cells were fixed and permeabilized according to the PE anti-mouse/rat Foxp3 staining set (eBioscience) and intracellularly stained for Foxp3 (eBioscience, FJK-16s) and CTLA4 (BD, UC10-4F10-11). The cells were acquired with the FACS Canto II (BD Pharmingen) and analyzed with FlowJo Version 8.87. Cells were usually pregated for a lymphocyte gate, singlets and living cells.

Lacher S.M., Bruttger J., Kalt B., Berthelet J., Rajalingam K., Wörtge S, & Waisman A. (2017). HMG-CoA reductase promotes protein prenylation and therefore is indispensible for T-cell survival. Cell Death & Disease, 8(5), e2824-.

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Intracellular Cytokine and Phospho-Protein Analysis

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Intracellular cytokine staining was performed as previously described [5 ]. Cell viability was determined with fixable viability dye eF660 or eF780 from eBioscience. The following antibodies were used: anti-IFN-γ (XMG1.2), anti-IL-17a (TC11-18H10.1), anti-CD4 (RM4–5), and anti-CD4 (GK1.5) (Biolegend). Cells from OT2 TCR^OVA recipients were stained with anti-CD45.2 (104)(Biolegend) and anti-CD45.1 (A20) (BD).
For phospho flow cytometry, cells were fixed for 7 min in 0.4% paraformaldehyde at room temperature and stained with fixable viability dye. After at an additional 7 mins in 4% paraformaldehyde the cells were permeablized in 90% methanol for 20 mins at 4°C. Cells were then stained with antibodies to pY701-STAT1 (58D6), pY705-STAT3 (D3A7), pS9-GSK3β (D85E12), pS473-AKT (D9E), pS235/236-S6 (D57.2.2E), Ku80 and/or β catenin (L54E2)(from Cell Signaling) and anti-CD4. Other reagents used as needed were goat anti-rabbit 488 (Jackson Immunoresearch), anti-RORγ(AFKJS-9, eBioscience), anti-RORγ (B2D) (eBioscience), anti-Tbet (4B10)(Biolegend). Tyramide amplification of pS473-AKT was performed using the kit (Molecular Probes). All samples were analyzed using a BD Facs Calibur or BD LSR2 and analyzed with FlowJo software.

McGuire D.J., Rowse A.L., Li H., Peng B.J., Sestero C.M., Cashman K.S., De Sarno P, & Raman C. (2014). CD5 ENHANCES TH17 DIFFERENTIATION BY REGULATING IFN-γ RESPONSE AND RORγT LOCALIZATION. European journal of immunology, 44(4), 1137-1142.

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Tetramer-based Detection of PyMT-specific T Cells

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PyMT peptides (MT_107-115, MT_241-250, MT_288-296) were folded into H-2D^q tetramers with human β2m and conjugated to PE. Tetramers were kindly provided by the NIH Tetramer Core Facility (Emory University, Atlanta, GA). Splenocytes from PyMT-vaccinated mice or MMTV-PyMT tumor bearing mice were harvested as described above. One million cells were stained with fixable viability dye (eF780, eBiosciences) in PBS for 30 minutes at 4ºC. Cells were washed and incubated with tetramers (1:100 dilution) for 30ʹ at room temperature, then incubated for 30ʹ at 4ºC with the following antibodies (eBiosciences): anti-mouse Fc block, anti-CD8α (eF450, clone 53–6.7), CD3ϵ (FITC, clone 145-2C11), CD62L (APC, clone MEL-14), CD44 (PE-Cy7, clone IM7). Additionally, for compensation in the tetramer+ PE channel, anti-CD8α PE was used. Flow cytometry data was acquired with a 3-laser S1200Ex flow cytometer (Stratedigm). Each experiment included single-stained compensation controls and fluorescence-minus one (FMO) staining for the PE channel. Compensation and data analysis were conducted with FlowJo v10 (Tree Star, Inc.).

DeVette C.I., Gundlapalli H., Lai S.C., McMurtrey C.P., Hoover A.R., Gurung H.R., Chen W.R., Welm A.L, & Hildebrand W.H. (2019). A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer. Oncoimmunology, 9(1), 1685300.

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Flow Cytometry Analysis of Tendon Cell Markers

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After harvest, tendon cells were washed twice in cell staining buffer (CSB) (BioLegend) and blocked for 15 minutes at room temperature (RT) in 20% human FcR blocking reagent (Miltenyi Biotech) diluted in CSB. All dead cells were excluded from analysis using fixable viability dye ef780 (1:1000 dilution) (eBioscience). Cells were stained in a buffer containing 20% FcR blocking reagent diluted in CSB at RT for 30 minutes. Antibody and isotype cocktails were prepared as indicated in Table 2. After washing, cells were fixed using Cytofix fixation buffer (BD Biosciences) for 20 minutes at RT. Flow cytometry was performed on a BD LSR Fortessa instrument calibrated daily with BD cytometer setup and tracking beads. Analysis of data was carried out using FlowJo software (Treestar).Table 2

Antibodies used for flow cytometry

Antibody	Catalogue number	Clone
PDPN Alexa Fluor 488 anti-human	337005 BioLegend	NC08
Alexa Fluor 488 Rat IgG2aκ isotype	400525 BioLegend	RTK2758
CD248 Alexa Fluor 647 anti-human	564994 BD Biosciences	B1/35
Alexa Fluor 647 mouse IgG1κ isotype	557714 BD Biosciences	MOPC-21
CD106 PE anti-human	305806 BioLegend	STA
PE mouse IgG1κ isotype	400112 BioLegend	MOPC-21
CD34 PerCP/Cy5.5 anti-human	343521 BioLegend	581
PerCP/Cy5.5 mouse IgG1κ isotype	400149 BioLegend	MOPC-21
CD45 BV605 anti-human	304041 BioLegend	H130
BV605 mouse IgG1κ isotype	400161 BioLegend	MOPC-21

All antibodies were diluted 1:50 for staining

Dakin S.G., Buckley C.D., Al-Mossawi M.H., Hedley R., Martinez F.O., Wheway K., Watkins B, & Carr A.J. (2017). Persistent stromal fibroblast activation is present in chronic tendinopathy. Arthritis Research & Therapy, 19, 16.

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PARP Isoform Expression in U87MG Cells

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Relative expression of PARP isoforms was determined using flow cytometry in U87MG cells after exposure to unlabelled olaparib. U87MG cells (1 × 10⁶ cells/well) were seeded in 96-well plates and exposed to olaparib (final concentration: 0–1 μM in 200 μL growth medium) for 3 h at 37 °C. Cells were washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA, 0.1% NaN₃) and centrifugation at 500 × g for 5 min. Immunostaining was performed using the Foxp3/transcription factor staining buffer set (eBioscience™, USA). Intracellular staining was conducted in permeabilisation buffer for 30 min at 4 °C in the dark separately using the following antibodies: AF488-conjugated anti-PARP-1 (1:100; sc-80070), AF594-conjugated anti-PARP-2 (1:100; sc-393310) and AF488-conjugated anti-PARP-3 (1:100; sc-390771) from Santa Cruz Biotechnology Inc. Fixable viability dye ef780 (1:4000; eBioscience™; 65-0865-14) was used for live and dead cells discrimination. Fixation of immunostained cells was performed for 15 min at room temperature. Flow cytometry was conducted on the CytoFLEX benchtop flow cytometer (Beckman Coulter, USA), with appropriate laser and filters, positive and negative controls. Data were analysed using FlowJo™ (Tree Star Inc., BD Biosciences, USA).

Chan C.Y., Hopkins S.L., Guibbal F., Pacelli A., Baguña Torres J., Mosley M., Lau D., Isenegger P., Chen Z., Wilson T.C., Dias G., Hueting R., Gouverneur V, & Cornelissen B. (2022). Correlation between molar activity, injection mass and uptake of the PARP targeting radiotracer [18F]olaparib in mouse models of glioma. EJNMMI Research, 12, 67.

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Flow Cytometric Analysis of Immune Cells

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We washed the cells with cold PBS and incubated them with fixable viability dye eF780 (eBioscience) at room temperature for 10 minutes. We then transferred the cells to V‐bottomed plates (Greiner Bio‐One) and allowed cells to incubate for 30 minutes at 4°C in the dark with the surface‐staining antibodies (Supplementary Table 3, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42319). Next, we washed the cells in fluorescence‐activated cell sorting buffer (1% bovine serum albumin and 0.1% sodium azide in PBS) and then fixed/permeabilized the cells for 30 minutes at 4°C in the dark with 100 μl fixation/permeabilization concentrate and diluent (catalog nos. 00‐5123‐43 and 00‐5223‐56, eBioscience); this step was followed by intracellular staining. After 60 minutes of staining at 4°C in the dark, cells were washed and measured on the BD LSRFortessa with 4 lasers (405, 488, 561, and 635 nm) using FACSDiva software version 8.0.1. We analyzed the resulting files using FlowJo.

Servaas N.H., Hiddingh S., Chouri E., Wichers C.G., Affandi A.J., Ottria A., Bekker C.P., Cossu M., Silva‐Cardoso S.C., van der Kroef M., Hinrichs A.C., Carvalheiro T., Vazirpanah N., Beretta L., Rossato M., Bonte‐Mineur F., Radstake T.R., Kuiper J.J., Boes M, & Pandit A. (2022). Nuclear Receptor Subfamily 4A Signaling as a Key Disease Pathway of CD1c+ Dendritic Cell Dysregulation in Systemic Sclerosis. Arthritis & Rheumatology (Hoboken, N.j.), 75(2), 279-292.

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Multiparametric Identification of MAIT Cells

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PBMCs from all donors were thawed and stained with fixable viability dye eF780 (eBioscience), after which the cells were stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8, CD45RO, CD161, CCR9, CXCR5, TCRVα7.2, IL-7Rα (CD127), and IL-18Rα (CD218α). For intracellular staining after overnight stimulation cells were fixed/permeabilized using Fixation/Permeabilization Concentrate and Diluent (Cat #00-5123-43, #00-5223-56, eBioscience) according to manufacturer’s protocol. In this panel IL-7Rα and IL-18Rα were replaced with IL-21 and IFN-γ. Details of used antibodies can be found in Supplementary Table 1. All samples were acquired on a BD LSRFortessa (BD Biosciences) using BD FACSDiva software v.8.0.1 (BD Biosciences). For data analysis FlowJoTM Software v10.8 (BD Life Sciences) was used. MAIT cells were defined using either CD161+ or IL-18Rα+, combined with TCRVα7.2+ CD3 cells. Gating of MAIT cells (CD161+ and IL-18Rα+ in combination with TCRVα7.2+) within CD4/CD8 populations is shown in Supplementary Figure 1.

Hinrichs A.C., Kruize A.A., Leavis H.L, & van Roon J.A. (2022). In patients with primary Sjögren’s syndrome innate-like MAIT cells display upregulated IL-7R, IFN-γ, and IL-21 expression and have increased proportions of CCR9 and CXCR5-expressing cells. Frontiers in Immunology, 13, 1017157.

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