Apc efluor 780

Manufactured by Thermo Fisher Scientific

Sourced in United States

The APC-eFluor 780 is a fluorescent dye used in flow cytometry applications. It is designed to provide a bright and stable signal for the detection of target cells or molecules. The dye has an excitation maximum at 650 nm and an emission maximum at 780 nm, making it suitable for use with common flow cytometry laser and filter configurations.

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CD4 APC-eFluor 780 (clone GK1.5, (2 citations) Anti-CD4-APC-eFluor 780 (SK3; (2 citations) Anti-mouse CD3 APC-eFluor 780 (2 citations) APC)–eFluor 780 (clone 30-F11 (2 citations) Anti-IFN-γ APC-eFluor 780 (2 citations) CD45RA APC-Alexa eFluor 780 (2 citations) APC-efluor® 780-conjugated CD3 (clone UCHT1; (2 citations) MHCII-APC-eFluor 780 (3 citations) Anti-CD45-APC-eFluor 780 (6 citations) Anti-B220-APC-eFluor 780 (2 citations)

35 protocols using apc efluor 780

Multiparameter Flow Cytometry Assay

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Antibodies against murine CD4 (RM4-5, AF700, BV650), CD8 (53-6.7, APC/Fire 750, BV785), CCR2 (SA203G11, BV421), CD11c (N418, APCCy7, Pe/Cy5), CD206 (C068C2, APC), CD11b (M1/70, BV421), F4/80 (BM8, BV510, BV711, FITC), TCRβ (H57-597, BV605), and NK1.1 (PK136, BV650) were purchased from BioLegend. Antibodies against CD4 (GK1.5, BUV395) and ST2 (U29-93, BV480) were purchased from BD Biosciences. Antibodies against B220 (RA3-6B2, AF488), CD11c (N418, e450, PE-Cy5.5), CD25 (PC61.5, PE-Cy7), CD4 (GK1.5, APC-eFluor 780, Super Bright 645), CD45 (30-F11, Pacific Orange), Foxp3 (FJK-16s, APC, e450, FITC), GITR (DTA-1, Super Bright 600), IL-10 (JES5-16E3, PE), KLRG1 (2F1, APC, APC-eFluor 780, PE-eFluor 610), NK1.1 (PK136, PE), and ST2 (RMST2-2, PE, PerCP-eFluor710) were purchased from eBioscience, Thermo Fisher Scientific. Viability dyes (e506, near IR, UV) were purchased from Invitrogen, Thermo Fisher Scientific.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.

Beppu L.Y., Mooli R.G., Qu X., Marrero G.J., Finley C.A., Fooks A.N., Mullen Z.P., Frias AB J.r., Sipula I., Xie B., Helfrich K.E., Watkins S.C., Poholek A.C., Ramakrishnan S.K., Jurczak M.J, & D’Cruz L.M. (2021). Tregs facilitate obesity and insulin resistance via a Blimp-1/IL-10 axis. JCI Insight, 6(3), e140644.

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Multiparametric Flow Cytometry of Immune Cells

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Single cell solutions of the spleen, kidneys, aorta, and perivascular adipose tissue (PVAT) were prepared to be stained with fluorophore-coupled antibodies for flow cytometric analyses. After blocking unspecific binding with Fc-block (Bio X Cell, USA), the following monoclonal antibodies were used for surface staining: CD45.2 (clone: 104, APC-eFluor780, eBioscience), CD90.2 (clone: 53-2.1, APC-eFluor780, eBioscience or PerCP, BioLegend), CD3 (clone: 145-2C11, PerCP, BD Biosciences), CD4 (clone: GK1.5, FITC, BioLegend or clone: RM4-5, V500, BD Biosciences), CD8 (clone: 53-6.7, PE-Cy7, eBioscience), CD11b (clone: M1/70, PE-Cy7, eBioscience), F4/80 (clone: BM8, APC, BioLegend), Ly6G (clone: 1A8, PE, BioLegend), and Ly6C (clone: AL-21, BD Biosciences).
Samples were acquired using the FACSCanto™ II (BD). Analysis was performed using the FlowJo Software (BD, USA).

Schüler R., Efentakis P., Wild J., Lagrange J., Garlapati V., Molitor M., Kossmann S., Oelze M., Stamm P., Li H., Schäfer K., Münzel T., Daiber A., Waisman A., Wenzel P, & Karbach S.H. (2019). T Cell-Derived IL-17A Induces Vascular Dysfunction via Perivascular Fibrosis Formation and Dysregulation of ·NO/cGMP Signaling. Oxidative Medicine and Cellular Longevity, 2019, 6721531.

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Comprehensive Immune Cell Profiling in Arthritis

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The isolation of cells from arthritic paws was performed as previously described⁶¹. Cell suspensions from lymph nodes, spleen, blood and paws were analyzed on a LSR II (BD Biosciences) upon staining with the following mAbs: anti-CD44-eFluor 450 (IM7), anti-CD4 PerCp (RM4-5), anti-PD-1 PE-Cy7 (J43), anti-CD45.2 APC-eFluor 780 (104), anti-CD11b PE-Cy7 (M1/70), anti-Ly-6C PerCp-Cy5.5 (HK1.4), anti-Ly-6G PE (1A8), anti-CD11c PE-Cy7 (N418), anti-CD45.1 APC (A20), anti-CD3e PE-Cy7 (145-2C11), anti-CD62L APC-Cy7 (Mel-14), anti-Fas PE (15A7) (all from eBioscience), anti-CD19 Alexa 488 (6D5) (Biolegend), anti-B220 Pacific Orange (RA3-6B2) (ThermoFisher Scientific), anti-I-Ab FITC (AF6-120.1) (BD Biosciences) and anti-CD11b biotinylated (MAC-1) grown in our laboratories. Staining of biotinylated Abs was followed by incubation with Pacific Orange labeled streptavidin (ThermoFisher Scientific). Expression of CXCR5 was assessed using anti-CXCR5 biotinylated (clone 2G8, BD Biosciences), 30 min at room temperature, followed by APC-eFluor 780 or Cy5 labeled streptavidin (eBioscience). Fluorescence-minus-one (FMO) or isotype control samples were used to assess background fluorescence and unspecific Ab binding. Data were analyzed with FACSDiva (BD Biosciences) and FlowJo Software (Treestar).

Moschovakis G.L., Bubke A., Friedrichsen M., Falk C.S., Feederle R, & Förster R. (2017). T cell specific Cxcr5 deficiency prevents rheumatoid arthritis. Scientific Reports, 7, 8933.

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Multiparameter Flow Cytometry Analysis

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All staining analyses were performed on an LSRII Fortessa flow cytometer using the following mAbs: anti–mouse CD3 (clone 145-2C11, hamster IgG1), CD4 (clone RM4-5, rat IgG2a), CD8 (clone 53–6.7, rat IgG2a), CD11b (clone M1/70, rat IgG2b), CD16/32 (clone 93, rat IgG2a), CD21 (clone 7G6, rat IgG2b), CD25 (clone PC61, rat IgG1), CD34 (clone RAM34, rat IgG2a), CD41 (clone MWReg30, rat IgG1), CD43 (clone S7, rat IgG2a), CD44 (clone IM7, rat IgG2b), CD45R/B220 (clone RA3-6B2, rat IgG2a), CD45.1 (clone A20, mouse IgG2a), CD45.2 (clone 104, mouse IgG2a), CD48 (clone HM48-1, Armenian hamster IgG), CD62L (clone MEL-14, rat IgG2a), CD117 (clone 2B8, rat IgG2b), CD127 (clone A7R34, rat IgG2a), CD135 (clone A2F10, rat IgG2a), CD150 (clone TC15-23F12,2, rat IgG2a), Ter119 (clone TER-119, rat IgG2b), Gr-1 (clone RB6-8C5, rat IgG2b), Sca-1 (clone E13-161,7, rat IgG2a), IgM (clone II/41, ratIgG2a), and Cxcr4 (clone 2B11, rat IgG2b). Antibodies were conjugated to biotin, BV 650, FITC, PE, APC, AF700, PE-cyanin (Cy) 5, PE-Cy7, eFluor 450, AF 647, APC-eFluor 780, peridinin chlorophyll protein PerCP-Cy5,5, or pacific blue and purchased from BD, eBioscience, BioLegend, or Sony. The lineage antibody cocktail included anti-CD3, anti-CD45R, anti-CD11b, anti-TER119, anti-CD41, and anti–Gr-1 mAbs. Secondary labeling was performed with a Streptavidin–pacific orange from Thermo Fisher Scientific.

Freitas C., Wittner M., Nguyen J., Rondeau V., Biajoux V., Aknin M.L., Gaudin F., Beaussant-Cohen S., Bertrand Y., Bellanné-Chantelot C., Donadieu J., Bachelerie F., Espéli M., Dalloul A., Louache F, & Balabanian K. (2017). Lymphoid differentiation of hematopoietic stem cells requires efficient Cxcr4 desensitization. The Journal of Experimental Medicine, 214(7), 2023-2040.

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Multiparametric Flow Cytometry Analysis of Immune Cell Phenotypes

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The following antibodies were used: anti-mouse TCR-β (H57-597) APC or Pacific Blue, PBS57 loaded CD1d tetramer APC or Pacific Blue, anti-mouse CD4 (GK1.5) PerCp-Cy5.5, anti-mouse CD8 (53-6.7) Am-cyan, anti-mouse NK1.1 (PK-136) PE-Cy7, anti-mouse CD44 (IM7) PerCp-Cy5.5, anti-mouse CD69 (H1-2F3) PE-Cy7, anti-mouse CD62L (MEL-14) eVolve605, anti-mouse IFN-γ (XMG1.2) FITC or APC, anti-mouse IL-4 (11B11) PE-Cy7, anti-mouse IL-17 (TC11-18H10) APC-eFluor780, anti-T-bet (eBio4B10) FITC, anti-RORγt (AFKJS-9) Pacific Blue, anti-GATA3 (L50-823) APC, and anti-PLZF (Mags-21F7) PE (all from eBioscience). Dead cells were excluded by staining with 1 µg/ml propidium iodide (Sigma-Aldrich). To measure intracellular cytokines, cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, Sigma-Aldrich) and ionomycin (1.5 µM, Sigma-Aldrich) in the presence of Monensin (3 µM, Sigma-Aldrich), permeabilized using Cytofix/Cytoperm Plus (BD), then stained with the appropriate antibodies. Transcription factor staining to identify committed cells was performed using the Foxp3/transcription factor staining kit (eBioscience) and intranuclear staining for T-bet, RORγt, GATA3 and PLZF. Data were acquired on a FACS Canto II (BD) and analyzed using FlowJo (TreeStar software ver. 9.9).

Kim Y.H., Kumar A., Chang C.H, & Pyaram K. (2017). Reactive Oxygen Species Regulate the Inflammatory Function of NKT Cells through Promyelocytic Leukemia Zinc Finger. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3478-3487.

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Comprehensive flow cytometry antibody panel

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The following antibodies were used for flow cytometric studies: Brilliant Violet 785™ anti-human CD3 Antibody (Biolegend®, clone OKT3, 317330), CD14 Monoclonal Antibody APC-eFluor 780 (eBioscience™, clone 61D3, 47-0149-42), PE/Cy7 anti-human CD15 (SSEA-1) antibody (Biolegend®, clone W6D3, 323029), CD19 Monoclonal Antibody PE (eBioscience™, clone HIB19, 12-0199-42). The following isotype controls were used: Mouse IgG1 kappa Isotype Control, APC-eFluor 780 (eBioscience™, clone P3.6.2.8.1, 47-4714-82), Brilliant Violet 785™ Mouse IgG2a, κ Isotype Ctrl Antibody (Biolegend®, MOPC-173, 400273), PE/Cy7 Mouse IgG1, κ Isotype Ctrl Antibody (Biolegend®, MOPC-21, 400125), Mouse IgG1 kappa Isotype Control, PE (eBioscience™, clone P3.6.2.8.1, 12-4714-81). All antibodies were used at 1/250 dilution.

Batalha I.L., Bernut A., Schiebler M., Ouberai M.M., Passemar C., Klapholz C., Kinna S., Michel S., Sader K., Castro-Hartmann P., Renshaw S.A., Welland M.E, & Floto R.A. (2019). Polymeric nanobiotics as a novel treatment for mycobacterial infections. Journal of Controlled Release, 314, 116-124.

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Multiparametric Flow Cytometry Analysis

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Cell surface and intracellular staining were performed using antibodies including CD3 (eFluor 450; eBioscience, Waltham, MA, United States; clone UCHT1), CD8a (FITC, eBioscience, clone RPA-T8; or APC-eFluor 780, eBioscience, clone RPA-T8), TRBV4-1 (PE; Miltenyi Biotec, Bergisch Gladbach, Germany; clone REA871), Ghost Dye (Violet 510; Tonbo Biosciences, San Diego, CA, United States), IFN-γ (PE-Cyanine7, eBioscience, clone 4S.B3), T-bet (BV421; BD Biosciences, San Jose, CA, United States; clone O4-46), Granzyme A (PE-Cyanine7, eBioscience, clone CB9), Granzyme B (FITC, BD eBioscience, clone GB11), Granzyme K (eFluor 660, eBioscience, clone G3H69), PRF1 (BV421, BD Biosciences, clone δG9), CCL4 (PerCP-eFluor 710, eBioscience, clone FL34Z3L), CCL5 (eFluor 660, eBioscience, clone VL1), and CXCR3 (PE-Cyanine7, eBioscience, clone CEW33D). Intracellular staining was performed on T cells stimulated with PMA (50 ng/mL; Sigma-Aldrich, St. Louis, MO, United States) and ionomycin (1 mM; Sigma-Aldrich) in the presence of GolgiStop (2/3 μL/mL, BD Biosciences) and Grid-plug (1 μL/mL, BD Biosciences) for 5 h. Flow cytometry was performed on a BD FACSCanto II flow cytometer using BD FACSDiva Software and FCS Express 5 software (De Novo Software, Los Angeles, CA, United States).

Shao M.M., Yi F.S., Huang Z.Y., Peng P., Wu F.Y., Shi H.Z, & Zhai K. (2022). T Cell Receptor Repertoire Analysis Reveals Signatures of T Cell Responses to Human Mycobacterium tuberculosis. Frontiers in Microbiology, 13, 829694.

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Immunostaining of Erythroid Progenitors

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Single-cell suspensions of freshly collected bone marrow cells were immunostained with APC-H7 (560185, BD Biosciences) or APCeFluor780 (eBioscience 47-1172-82) -conjugated rat anti-KIT, FITC-conjugated rat anti-CD71 (553266, BD Biosciences), APC-conjugated rat anti-Ter119 (557909, BD Biosciences) and PE-conjugated streptavidin (554061, BD Biosciences) and biotin-conjugated rat anti-CD123 (555070, BD Biosciences) antibodies for the detection of erythroid progenitor (CFU-E) cells, as previously described [52 (link)]; FITC-conjugated rat anti-CD11b (553310, BD Biosciences), PerCP-Cy^TM5.5-conjugated rat anti-mouse CD19 (45-0193-82, eBioscience) and PE-conjugated rat anti-mouse CD4 (553049, BD Biosciences), and APC-conjugated rat anti-myeloid CD8 (553035, BD Biosciences) and PE-Cy^TM7- conjugated rat anti-myeloid B220 (25-0452-82, eBioscience) antibodies were used for the detection of freshly collected bone marrow cells. Stained cells were analysed with a BD Accuri C6 or a Fortessa analyser (BD Biosciences), and FlowJo (Tree Star) was used for data acquisition and analysis.
Additional details of the methods are included in the supplementary data.

Pawlikowska P., Delestré L., Gregoricchio S., Oppezzo A., Esposito M., Diop M.B., Rosselli F, & Guillouf C. (2023). FANCA deficiency promotes leukaemic progression by allowing the emergence of cells carrying oncogenic driver mutations. Oncogene, 42(37), 2764-2775.

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Humanized Immune System Engraftment in NRG Mice

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NOD-Rag1null IL2rgnull (NRG) mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). NRG pups (24–72 h old) were irradiated twice with 3 cGy 3 h apart and then engrafted intrahepatically with 10⁵–10⁶ CD34⁺ hematopoietic stem cells (HSC) isolated from human umbilical cord blood. At 12 weeks post engraftment, blood was collected to quantify human immune cell reconstitution using flow cytometry. Erythrocytes were lysed using an ACK lysis buffer and the remaining cells were treated with both anti-human Fc Receptor Binding Inhibitor and anti-mouse CD16/CD32 antibodies (eBiosciences). Cells were then stained with an antibody cocktail (mCD45-AlexaFluor 700 (1:50), hCD45-Pacific Blue (1:20), hCD3e-Qdot 605 (1:100), hCD4-PerCP-Cy5.5 (1:20), hCD8a-PE-Cy7 (1:20)), followed by fixable viability dye (APC-eFluor 780; eBiosciences). Samples were run on the Cytoflex LX flow cytometer equipped with a flow rate calibrator and analyzed using FlowJo software version 10 (Tree Star, Ashland, OR, USA). Mice with at least 10% or 50,000 per mL hCD45+ leukocytes in the blood were selected for subsequent experiments.

Afkhami S., D’Agostino M.R., Vaseghi-Shanjani M., Lepard M., Yang J.X., Lai R., Choi M.W., Chacon A., Zganiacz A., Franken K.L., Ertl H.C., Ottenhoff T.H., Jeyanathan M., Gillgrass A, & Xing Z. (2023). Intranasal multivalent adenoviral-vectored vaccine protects against replicating and dormant M.tb in conventional and humanized mice. NPJ Vaccines, 8, 25.

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Isolation and Identification of Kidney Immune Cells

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Briefly, kidneys were collected, minced, and incubated with DNase I (90083, ThermoFisher Scientific) and type IV collagenase (sigma, 11088858001) in RPMI 1640 at room temperature in a shaker for 30 min. The digestion was ended using RPMI and FBS (085-150, WISENT). The tissue suspension was milled into a single-cell suspension through a mesh with a 40 μm pore size. After blocking, the fresh suspensions were incubated with anti-mouse CD45 antibody (30-F11, 47-0451-82, APC-eFluor™ 780, eBioscience™) to ensure the number of immune cells. Then, the suspensions were labeled with Ly-6G/Ly-6C antibody (RB6-8C5, 11-5931-82, FITC eBioscience™) and anti-mouse F4/80 antibody (BM8, 48-4801-82, PE, eBioscience™) to identify neutrophils and macrophages, respectively.

Wu F., Chen C., Lin G., Wu C., Xie J., Lin K., Dai X., Chen Z., Ye K., Yuan Y., Chen Z., Ma H., Lin Z, & Xu Y. (2024). Caspase-11/GSDMD contributes to the progression of hyperuricemic nephropathy by promoting NETs formation. Cellular and Molecular Life Sciences: CMLS, 81(1), 114.

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