Anti cd8 apc efluor780

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD8-APC-eFluor780 is a fluorescently-labeled monoclonal antibody that binds to the CD8 cell surface antigen. It is designed for use in flow cytometry applications.

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Close spelling variants of this product

Anti-CD8 (106 citations) CD8-APC (44 citations) Anti-CD8-APC (40 citations) APC-eFluor780–conjugated anti-CD8 (2 citations)

16 protocols using anti cd8 apc efluor780

Multiparameter Flow Cytometry Profiling

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Antibodies from eBiosciences included anti-CD8-APC-eFluor780 (47-0081), anti-CD44-APC (17-0441-81), anti-CD69-PERCPcy5.5 (45-0691), anti CD98-PE (12-0981-81; eBiosciences), and anti CD62L-PEcy7 (25-0621). Anti-CD4-BD605NC was acquired from BD Biosciences (San Jose, CA). Cells were stained for 20 minutes at 4°C in PBS, 5% BSA, 0.1% NaN3. Cell proliferation was assessed using Cell Trace Violet (C34557; Life Technologies) according to the manufacturer’s recommendations. Samples were acquired on a BD LSRII flow cytometer and analyzed with Treestar FlowJo software. Cell cycle analysis was performed using BD APC BrdU Flow kit per the manufacturer’s instructions (552598; BD Biosciences). Cells were sorted on a MoFlow (Beckman-Coulter) or Reflection (i-Cyt).

Verbist K.C., Guy C.S., Milasta S., Liedmann S., Kamiński M.M., Wang R, & Green D.R. (2016). Metabolic Maintenance of Cell Asymmetry following Division in Activated T Lymphocytes. Nature, 532(7599), 389-393.

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Multiparametric Flow Cytometry Analysis of Regulatory T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis Shield, Oslo, Norway) centrifugation and stored in liquid nitrogen until further use. Cells were defrozen and subsequently fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) and stained for flow cytometric analysis with anti-CD3-PerCP (BD Biosciences, San Jose, CA, USA), anti-CD8-APC-eFluor780 (eBioscience), anti-CD127-Alexa647 (Biolegend, San Diego, CA, USA), anti-CD25-PE (eBioscience), anti-CD45RO-Alexa700 (Biolegend), anti-CD45RA-Alexa605 (BD Biosciences) and anti-FoxP3-Alexa488 (eBioscience). Cells were measured on an LSR-II flow cytometer (BD Biosciences) and the data were analyzed with Kaluza 1.2 Analysis Software (Beckman Coulter, Woerden, The Netherlands).
Absolute numbers of lymphocytes in EDTA anti-coagulated blood were determined using the BD MultiTest TruCount method with sixcolor MultiTest reagents detecting CD45, CD3, CD4, CD8, CD19, CD16 and CD56 (BD Biosciences) according to the manufacturer’s instructions. Percentages of (functional) Treg subsets were characterized as described before [7 (link),16 (link)] and CD4+ T-cell counts were used to calculate the absolute numbers of these subsets.

Janssen K.M., Westra J., Chalan P., Boots A.M., de Smit M.J., van Winkelhoff A.J., Vissink A, & Brouwer E. (2016). Regulatory CD4+ T-Cell Subsets and Anti-Citrullinated Protein Antibody Repertoire: Potential Biomarkers for Arthritis Development in Seropositive Arthralgia Patients?. PLoS ONE, 11(9), e0162101.

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Multiparametric Flow Cytometry of Immune Cell Subsets

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Single cell suspensions of total splenocytes, enriched B cells or BM were stained with different combinations of the following antibodies: Anti-CD4 APC-eFluor 780, anti-CD8 APC-eFluor 780, anti-Gr1 APC-eFluor 780, anti-F4/80 APC-eFluor 780, anti-B220 APC, anti-B220 APC-eFlour 780, anti-B220 FITC, anti CD19 PeCy7, anti-CD38 Alexa Fluor 700, anti-CD93 APC, anti-IgM PerCP-eFluor 710, anti CD21/CD35 eFluor 450 (eBiosciences), anti-CD23 PE (BioLegend), anti-CD4 PE-CF594, anti-CD8 PE-CF594, anti-Ly-6G and Ly-6C PE-CF594 and anti-IgG1 BV421 (BD biosciences). Live dead aqua stain was added to separate dead cells (Life Technologies) and eOD-GT8-specific cells were visualized by the addition of FITC-conjugated eOD-GT8 and PE-conjugated eOD-GT8 CD4bs knock-out. BG505 SOSIP- (Sok et al., 2014 (link)) and 2cc-specific memory B cells were visualized by the addition of biotinylated protein with the addition of streptavidin conjugated PE and APC respectively (BD Biosicences).

Dosenovic P., von Boehmer L., Escolano A., Jardine J., Freund N.T., Gitlin A.D., McGuire A.T., Kulp D.W., Oliveira T., Scharf L., Pietzsch J., Gray M.D., Cupo A., van Gils M.J., Yao K.H., Liu C., Gazumyan A., Seaman M.S., Björkman P.J., Sanders R.W., Moore J.P., Stamatatos L., Schief W.R, & Nussenzweig M.C. (2015). Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knock-In Mice. Cell, 161(7), 1505-1515.

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Neoantigen-Specific T-cell Response Quantification

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The neoantigen-specific T-cell response was determined using intracellular cytokine staining (ICS) performed by FC detection and IFNγ ELIspot as previously described^{19 (link)} For the FC analysis, the following antibodies were utilized according to different panels: anti-CD3-Alexafluor488 (cat. 53–0031-82, eBioscience), anti-CD3-PEefluor610 (cat. 61–0031-82, eBioscience), anti-CD4-PerCP-Cy5.5 (cat. 45–0042-82, eBioscience), anti-CD8-APCeFluor780 (cat 47–0081-82, eBioscience), anti-CD45-efluor450 (cat. 48–0451-82, eBioscience), anti-CD45-APC (cat. 559,864, BD), NK1.1-BV786 (cat. 740,853, Bio Legend), anti-CD11b-FITC (cat. 53,310, BD), anti-CD11c-SB645 (cat. 64–0114-82, Ebioscience), anti-LY6C-PE-Cy7 (cat. 560,593, BD), anti-LY6G-Alexafluor700 (cat. 561,236, BD), anti-IFN-γ-PE (cat. 12–7311-82, eBioscience), anti-TNF-α-PE-Cy7 (cat. 25–7321-82, eBioscience), anti-TNF-α-eFluor450 (cat. 48–7321-82, eBioscience FC was carried out with a Citoflex flow cytometer (Beckman Coulter) and data analyzed with Cytexpert software (Beckman Coulter). For the IFNγ ELIspot cells were plated at 4 × 10⁵ and 2 × 10⁵ cells/well in duplicate and spots were counted using an automated ELISPOT reader (Aelvis ELIspot reader, A.EL.VIS Gmbh, Germany).

Lione L., Salvatori E., Petrazzuolo A., Massacci A., Maggio R., Confroti A., Compagnone M., Aurisicchio L., Ciliberto G, & Palombo F. (2021). Antitumor efficacy of a neoantigen cancer vaccine delivered by electroporation is influenced by microbiota composition. Oncoimmunology, 10(1), 1898832.

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Multiparameter Flow Cytometry of PBMCs

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Peripheral blood samples were drawn from all participants on the day of inclusion, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using lymphocyte separation medium (Corning, Manassas, VA, USA). The isolated PBMCs were cryopreserved in liquid nitrogen until use.
Cryopreserved PBMCs were thawed and stained with Live/Dead Fixable Red Stain dye (Life Technologies, Gaithersburg, MD, USA) and fluorochrome-conjugated monoclonal antibodies for cell surface proteins. The stained cells were analyzed using an LSR II instrument (BD Biosciences, San Jose, CA, USA) and FlowJo v10 software (FlowJo, Ashland, OR, USA). The following antibodies were used: anti-CD3-V500 (BD Biosciences), anti-CD4-BV711 (BD Biosciences), anti-CD8-APC-eFluor780 (eBioscience, San Diego, CA, USA), anti-CCR5-BV650 (BD Biosciences), anti-CXCR3-PE (R&D Systems, Minneapolis, MN, USA), anti-CCR6-BV786 (BD Biosciences), anti-CCR8-PE (R&D Systems), anti-CRTh2-PerCP/Cy5.5 (Biolegend, San Diego, CA, USA), anti-CCR4-PE-Cy7 (BD Biosciences), anti-CCR3-APC (R&D systems), anti-CCR10-PE (R&D systems), anti-CCR9- PerCP/Cy5.5 (Biolegend), and anti-CX₃CR1-APC (Biolegend).

Choi Y.J., Lim D., Byeon S.H., Shin E.C, & Chung H. (2022). Chemokine Receptor Profiles of T Cells in Patients with Age-Related Macular Degeneration. Yonsei Medical Journal, 63(4), 357-364.

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Comprehensive Murine Immune Cell Profiling

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Mouse cells were preincubated with purified antimouse CD16/CD32, and human cells were incubated with FcR-blocking reagent (BD Biosciences). Isolated cells were stained with labeled antibodies in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA). Dead cells were excluded based on staining with Live/Dead fixable dye (eBioscience). Forkhead box P3 (FOXP3) fixative solution (eBioscience) was used for FOXP3 staining. Prepared samples were analyzed using a flow cytometer (FACSCalibur or FACSAria; BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The following beads and antibodies were used: OneComp eBeads (eBioscience), antimouse CD11c Brilliant Violet 421 (BioLegend), antimouse CD11b PE-cy7 (eBioscience), GR1-APC (eBioscience), LY6C-APC-eFluor (eBioscience), F4/80 PE (eBioscience), NK-1.1 Percp-Cy5.5 (eBioscience), anti-CD45 FITC (eBioscience), Live/Dead Aqua (eBioscience), anti-CD4 eFluor 450 (eBioscience), anti-CD8 APC eFluor 780 (eBioscience), anti-CD3 Percp-Cy5.5 (eBioscience), anti-CD19 PE-CY7 (eBioscience), anti-CD25 APC (eBioscience), anti-Foxp3 PE (eBioscience), and anti-CD3 APC (BioLegend).

Zhang H., Jiang R., Zhou J., Wang J., Xu Y., Zhang H., Gu Y., Fu F., Shen Y., Zhang G., Feng L., Zhang X., Chen Y, & Shen F. (2020). CTL Attenuation Regulated by PS1 in Cancer-Associated Fibroblast. Frontiers in Immunology, 11, 999.

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Multiparameter Flow Cytometry Profiling

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Isolation and Activation of CD4+ and CD8+ T Cells

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CD4⁺ and CD8⁺ T cells were isolated from total splenocytes using the EasySep^TM Mouse CD4⁺ T Cell Isolation Kit and the EasySep^TM Mouse CD8⁺ T cell Isolation Kit, respectively, according to the manufacturer’s protocol (Stemcell). 5 μg/mL anti-CD3 (eBioscience; 145-2C11) and 2 μg/mL anti-CD28 (eBioscience; 37.51) were co-immobilized on 24-well plates overnight at 4 C° and wells were washed 3 times with PBS prior to T cell stimulation. 1.5 × 10⁶ CD4⁺ or CD8⁺ T cells were incubated at 37 °C for 48 h in RPMI supplemented with 10% FBS (Invitrogen/Wisent), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (Wisent/Sigma), with or without 1 μM bpV(phen). Cells were collected and incubated for an additional 24 h in the absence of anti-CD3 and CD28 in RPMI with or without 1 μM bpV(phen). Cells were counted and labelled with PerCP-Cy5.5 anti-CD4 (eBioscience; RM4-5), APC-eFluor780 anti-CD8 (eBioscience; 53–6.7), and APC anti-CXCR3 (eBioscience; CXCR3-173). Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2.

Van Den Ham K.M., Smith L.K., Richer M.J, & Olivier M. (2017). Protein Tyrosine Phosphatase Inhibition Prevents Experimental Cerebral Malaria by Precluding CXCR3 Expression on T Cells. Scientific Reports, 7, 5478.

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Isolation and Characterization of Brain-Infiltrating Immune Cells

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Brains were digested in RPMI containing 1.6 mg/mL collagenase (type IV; Sigma-Aldrich) and 200 µg/mL DNase I (Sigma-Aldrich) at 37 °C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare) and debris was filtered out using a 70 µm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). Cells were labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), PerCP-Cy5.5 anti-CD4 (eBioscience; RM4–5) and APC-eFluor780 anti-CD8 (eBioscience; 53–6.7). Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2. Mice were perfused for the analysis of brain sequestered cells.

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Isolation and Characterization of B Cells

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Frozen lymph node (LN) cell suspensions collected from macaques vaccinated with VLP-RC1–4fill^{12 (link)} were thawed and incubated in FACS buffer (1xPhosphate-buffered saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti human antibodies at 1:200 dilution: anti CD3-APC-eFluor 780 (Invitrogen, 47-0037-41), anti CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), anti CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti CD20-PE-Cy7 (BD biosciences, 335793), and anti CD38 FITC (Stem Cell, 60131FI) and the LIVE/DEAD marker (Biolegend, 77184) at 1:400 dilution. Avi-tagged and biotinylated baits conjugated to fluorophore labeled streptavidin were added to the antibody mixture and incubated with the LN cells for 30 min. Single B cells were sorted into individual wells of a 96-well plates containing 5 μl of TCL lysis buffer (Qiagen, 1031576) and 1% beta-mercaptoethanol per well using a FACS Aria III (Becton Dickinson). The cell lysates were stored at −80°C or immediately used for subsequent mRNA purification.
To interrogate the B cell receptor specificity of the retrogenic B cells, the transduced B cells were incubated with Avi-tagged-biotinylated RC1 in complex with fluorophore labeled streptavidin for 30 min on ice. Alternatively, cells were incubated with fluorophore labeled streptavidin in the absence of RC1 for 30 min on ice.

Wang Z., Merkenschlager J., Chen S.T., Oliveira T.Y., Ramos V., Gordon K.M., Yao K.H., Jankovic M., Gazumyan A., Nussenzweig M, & Escolano A. (2019). Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques. Journal of immunological methods.

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