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Anti cd3 apc efluor780

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The Anti-CD3-APC-eFluor780 is a fluorescently-labeled antibody product designed for use in flow cytometry applications. It binds to the CD3 receptor expressed on the surface of T cells. The antibody is conjugated to the APC-eFluor780 fluorescent dye, which can be detected using appropriate instrumentation.

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Lab products found in correlation

Anti cd20 pecy7, by BD (4 mentions) Anti cd14 apc efluor 780, by Thermo Fisher Scientific (4 mentions) Anti cd16 apc efluor 780, by Thermo Fisher Scientific (4 mentions) Anti cd8 apc efluor780, by Thermo Fisher Scientific (4 mentions) Pan b cell isolation kit, by Miltenyi Biotec (3 mentions) Rnasin ribonuclease inhibitor, by Promega (3 mentions) Zombie nir, by BioLegend (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Anti cd8 vioblue, by Miltenyi Biotec (1 mentions) 7 aad solution, by BD (1 mentions) 96 well plate, by Greiner (1 mentions) Macsquant flow cytometer, by Miltenyi Biotec (1 mentions) Lsr fortessa 2, by BD (1 mentions) Live dead reagent, by Thermo Fisher Scientific (1 mentions) Anti hla dr bv650, by BioLegend (1 mentions) Facsdiva software, by BD (1 mentions) Tcl lysis buffer, by Qiagen (1 mentions) Facsaria 3, by BD (1 mentions) Anti cd38 fitc, by STEMCELL (1 mentions) Anti cd4 fitc, by BioLegend (1 mentions)

Close spelling variants of this product

Anti-CD3 (512 citations) CD3-APC (57 citations) Anti-CD3-APC (40 citations) CD3-APC-eFluor780 (14 citations) Anti-human CD3/APC-eFluor780 (3 citations)

14 protocols using anti cd3 apc efluor780

1

Quantifying CD69 Expression in Jurkat T Cells

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J.RT3-T3.5 2B9 cells (1 million) were transduced by direct addition of 1 mL HEK293T supernatant (see S1 Methods) and incubated for 72 h. Transduced J.RT3-T3.5 2B9 cells and co-cultures of these cells with antigen-presenting cells (100,000 cells each) were incubated in 96-well plates (Greiner Bio-one) for approximately 16 h. Supernatants containing Jurkat cells were removed and the cells pelleted. Cells were washed once with PBS with 0.1% fetal bovine serum (Gibco) before incubation with anti-CD69-PE (12-0699-42, Thermo Fisher Scientific), anti-CD3-APC-eFluor780 (47-0038-42, Thermo Fisher Scientific), and anti-CD8-VioBlue (130-110-684, Miltenyi Biotec) for 30 min at 4°C. Cells were pelleted and washed once with PBS with 0.1% fetal bovine serum. Cells were pelleted and resuspended in 7-AAD solution (51–6898, BD Pharmingen) before data were acquired on a Miltenyi MACSQuant flow cytometer. Data were analyzed using FlowJo v10.6.0 (FlowJo LLC). CD69-positive events were quantified upon gating on lymphocytes (SSC-A vs FSC-A), singlets (FSC-A vs FSC-H), live cells (7-AAD vs FSC-A), and CD8- and CD3-positive cells (S1 Fig).
Hiscox M.J., Wasmuth A., Williams C.L., Foot J.N., Wiedermann G.E., Fadda V., Boiani S., Cornforth T.V., Wikiert K.A., Bruton S., Cartwright N., Anderson V.E., Barnes C.S., Vieira J.V., Birch-Machin I., Gerry A.B., Miller K, & Pumphrey N.J. (2024). Selection, engineering, and in vivo testing of a human leukocyte antigen–independent T-cell receptor recognizing human mesothelin. PLOS ONE, 19(4), e0301175.
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2

Single-cell isolation of SARS-CoV-2 reactive B cells

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B cells from PBMCs of uninfected controls or of COVID-19 convalescent individuals were enriched using the pan-B-cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were subsequently stained in FACS buffer (PBS + 2% FCS + 1mM EDTA) with the following antibodies/reagents (all at 1:200 dilution) for 30 min on ice: anti-CD20-PE-Cy7 (BD Biosciences, 335828), anti-CD14-APC-eFluor 780 (Thermo Fischer Scientific, 47-0149-42), anti-CD16-APC-eFluor 780 (Thermo Fischer Scientific, 47-0168-41), anti-CD3-APC-eFluor 780 (Thermo Fischer Scientific, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), Zombie NIR (BioLegend, 423105), as well as fluorophore-labeled ovalbumin (Ova) and peptides. Live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−peptide-PE+peptide-AF647+ B cells were single-cell sorted into 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors [Promega, N2615]) per well using a FACS Aria III, and the analysis was performed with FlowJo software. The isolation of SD1-enriched B cells was performed similarly, except that sorted cells were live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−RBD−SD1-RBD-PE+SD1-RBD-AF647+. The gating strategy is shown in fig. S1C.
Bianchini F., Crivelli V., Abernathy M.E., Guerra C., Palus M., Muri J., Marcotte H., Piralla A., Pedotti M., De Gasparo R., Simonelli L., Matkovic M., Toscano C., Biggiogero M., Calvaruso V., Svoboda P., Rincón T.C., Fava T., Podešvová L., Shanbhag A.A., Celoria A., Sgrignani J., Stefanik M., Hönig V., Pranclova V., Michalcikova T., Prochazka J., Guerrini G., Mehn D., Ciabattini A., Abolhassani H., Jarrossay D., Uguccioni M., Medaglini D., Pan-Hammarström Q., Calzolai L., Fernandez D., Baldanti F., Franzetti-Pellanda A., Garzoni C., Sedlacek R., Ruzek D., Varani L., Cavalli A., Barnes C.O, & Robbiani D.F. (2022). Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2. bioRxiv.
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3

Comprehensive Immunophenotyping of T Cells

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Cells were stained with LIVE/DEAD reagent (Invitrogen, MA, USA), anti-CD3 APC-eFluor780 (ThermoFisher, MA, USA), anti-CD4-BUV395, anti-CD8-BV786, anti-CD27-BUV737, anti-CD45RA-APC-H7 and anti-HLADR-BV650 (BioLegend, CA, USA), anti-CD161-PE, and anti-ϒδ TCR-PE-CY7 (BD Life Sciences, NJ, USA) and anti-Vδ1-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) (see Supplemental Table 1). Data were acquired on an LSR-Fortessa II, and BD FACSDiva Software (BD Life Sciences, NJ, USA).
Karunathilaka A., Halstrom S., Price P., Holt M., Lutzky V.P., Doolan D.L., Kupz A., Bell S.C., Thomson R.M., Miles J.J, & Ratnatunga C.N. (2022). CD161 expression defines new human γδ T cell subsets. Immunity & Ageing : I & A, 19, 11.
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4

Enrichment and Single-Cell Sorting of SARS-CoV-2 Reactive B Cells

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B cells from PBMCs of uninfected controls or of COVID-19 convalescent individuals were enriched using the pan-B-cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec, 130–101-638). The enriched B cells were subsequently stained in FACS buffer (PBS + 2% FCS + 1 mM EDTA) with the following antibodies/reagents (all at 1:200 dilution) for 30 min on ice: anti-CD20-PE-Cy7 (BD Biosciences, 335828), anti-CD14-APC-eFluor 780 (Thermo Fischer Scientific, 47–0149-42), anti-CD16-APC-eFluor 780 (Thermo Fischer Scientific, 47–0168-41), anti-CD3-APC-eFluor 780 (Thermo Fischer Scientific, 47–0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47–0086-42), Zombie NIR (BioLegend, 423105), as well as fluorophore-labeled ovalbumin (Ova) and peptides. Live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−peptide-PE+peptide-AF647+ B cells were single-cell sorted into 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors [Promega, N2615]) per well using a FACS Aria III, and the analysis was performed with FlowJo software. The isolation of SD1-enriched B cells was performed similarly, except that sorted cells were live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−RBD−SD1-RBD-PE+SD1-RBD-AF647+. The gating strategy is shown in fig. S1C.
Bianchini F., Crivelli V., Abernathy M.E., Guerra C., Palus M., Muri J., Marcotte H., Piralla A., Pedotti M., De Gasparo R., Simonelli L., Matkovic M., Toscano C., Biggiogero M., Calvaruso V., Svoboda P., Cervantes Rincón T., Fava T., Podešvová L., Shanbhag A.A., Celoria A., Sgrignani J., Stefanik M., Hönig V., Pranclova V., Michalcikova T., Prochazka J., Guerrini G., Mehn D., Ciabattini A., Abolhassani H., Jarrossay D., Uguccioni M., Medaglini D., Pan-Hammarström Q., Calzolai L., Fernandez D., Baldanti F., Franzetti-Pellanda A., Garzoni C., Sedlacek R., Ruzek D., Varani L., Cavalli A., Barnes C.O, & Robbiani D.F. (2023). Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein. Science Immunology.
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5

Single-cell sorting of B cells

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B cells from PBMCs of uninfected controls or of COVID-19 convalescent individuals were enriched using the pan-B-cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were subsequently stained in FACS buffer (PBS + 2% FCS + 1mM EDTA) with the following antibodies/reagents (all at 1:200 dilution) for 30 min on ice: anti-CD20-PE-Cy7 (BD Biosciences, 335828), anti-CD14-APC-eFluor 780 (Thermo Fischer Scientific, 47-0149-42), anti-CD16-APC-eFluor 780 (Thermo Fischer Scientific, 47-0168-41), anti-CD3-APC-eFluor 780 (Thermo Fischer Scientific, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), Zombie NIR (BioLegend, 423105), as well as fluorophore-labeled ovalbumin (Ova) and peptides. Live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−peptide-PE+peptide-AF647+ B cells were single-cell sorted into 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors [Promega, N2615]) per well using a FACS Aria III, and the analysis was performed with FlowJo software. The isolation of SD1-enriched B cells was performed similarly, except that sorted cells were live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−RBD−SD1-RBD-PE+SD1-RBD-AF647+. The gating strategy is shown in fig. S1C.
Bianchini F., Crivelli V., Abernathy M.E., Guerra C., Palus M., Muri J., Marcotte H., Piralla A., Pedotti M., De Gasparo R., Simonelli L., Matkovic M., Toscano C., Biggiogero M., Calvaruso V., Svoboda P., Cervantes Rincón T., Fava T., Podešvová L., Shanbhag A.A., Celoria A., Sgrignani J., Stefanik M., Hönig V., Pranclova V., Michalcikova T., Prochazka J., Guerrini G., Mehn D., Ciabattini A., Abolhassani H., Jarrossay D., Uguccioni M., Medaglini D., Pan-Hammarström Q., Calzolai L., Fernandez D., Baldanti F., Franzetti-Pellanda A., Garzoni C., Sedlacek R., Ruzek D., Varani L., Cavalli A., Barnes C.O, & Robbiani D.F. (2023). Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein. Science Immunology.
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6

Isolation and Characterization of B Cells

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Frozen lymph node (LN) cell suspensions collected from macaques vaccinated with VLP-RC1–4fill12 (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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7

Quantification of Cross-Reactive T Cells

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This procedure was performed using modified versions of protocols described previously (12 , 13 (link)). Briefly, human PBMCs were stimulated with 17.7 μg/ml of TT lysate (MassBiologics, University of Massachusetts Medical School, MA) for 5-7 days. Spleen cells from DR3 mice immunized with protein or peptide were harvested 10-14 days post immunization. Human PBMCs were stained with 1 μg of DR3 tetramer for 3-4 hours at 37°C; and mouse spleen cells were stained for 2 hours. After tetramer staining, the cells were washed in PBS containing 2% FCS (FACS buffer) and surface markers were detected by anti-CD4-FITC (Biolegend, San Diego, CA) and anti-CD3-APCeFluor™780 (Invitrogen, Waltham, MA) for human cells and anti-CD4-FITC and anti-CD3-APC/Fire™750 (Biolegend, San Diego, CA) for mouse cells on ice for 30 minutes after Fc blocking. Cells were analyzed by the BD LSR Fortessa™ Cell Analyzer (BD, Franklin Lakes, NJ). When determining the percentage of single epitope reactive T cells from dual tetramer staining as seen in Figure 6, for SmD66-80, the percentage of single SmD66-80 tetramer positive T cells was the sum of cells in Q1 and Q2; for the ABC247-261 Mimic and TT peptides, the percentage of single Clostridium tetani epitope tetramer positive T cells was the sum of cells in Q2 and Q3. Cross-reactive T cells between SmD66-80 and Clostridium tetani epitopes were detected in Q2.
Zhao Z., Anderson A.N., Kannapell C.C., Kwok W.W., Gaskin F, & Fu S.M. (2022). HLA-DR3 restricted environmental epitopes from the bacterium Clostridium tetani have T cell cross-reactivity to the SLE-related autoantigen SmD. Frontiers in Immunology, 13, 928374.
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8

Flow Cytometric Analysis of Activated CD4+ T Cells

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Activated CD4+ T cells were stained with the following mAb: anti-CD3-APC-eFluor780, anti-CD4-APC, anti-CD25-PE, anti-Foxp3-Pac blue (eBioscience), and anti-pS6 (Ser235)-FITC (Cell Signaling Technology) using buffers from eBioscience. The stained cells were analyzed on a LSR II flow cytometer and data were analyzed with the Flowjo software package.
Hawse W.F., Sheehan R.P., Miskov-Zivanov N., Menk A.V., Kane L.P., Faeder J.R, & Morel P.A. (2015). Differential regulation of PTEN by TCR, Akt and FoxO1 controls CD4+ T cell fate decisions. Journal of immunology (Baltimore, Md. : 1950), 194(10), 4615-4619.
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9

Quantifying Chemokine Receptor Expression

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Vehicle- and PTX-treated cells were separately stained with anti-CCR6, followed by anti-mouse IgG-APC as previously described. Cells were washed two times in Labelling Buffer. Vehicle-treated cells were stained with anti-CD3 APC-eFluor780 (eBioscience, clone UCHT1, Hatfield, UK) and PTX-treated cells were stained with anti-CD3 AlexaFluor700 (eBioscience, clone UCHT1) diluted in Labelling Buffer and incubated for 15 minutes at 37°C. Cells were washed three times in RT HBSS. An equivalent number (determined by Trypan Blue Stain) of vehicle- and PTX-treated cells were combined to a final density of 1x106 cells/ml and loaded with 1 μM Fura Red, AM as previously described.
Wendt E.R., Ferry H., Greaves D.R, & Keshav S. (2015). Ratiometric Analysis of Fura Red by Flow Cytometry: A Technique for Monitoring Intracellular Calcium Flux in Primary Cell Subsets. PLoS ONE, 10(4), e0119532.
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10

Multiparameter Analysis of CD8+ T Cells

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533IYSTVASSL541 (IVL533-541)-tetramer was bought from MBL (cat. NO.TS-M520-1), anti-CD8α depletion antibody (clone: YTS 169.4). Rat IgG2b isotype antibody (clone: LTF-2) was purchased from Bio X Cell. Antibodies for flow cytometry included anti-CD3-APC-eFluor780 (clone: 17A2; eBioscience), anti-CD4-Pacific Blue (clone: GK1.5; BioLegend), anti–CD8α-PerCP/Cy5.5 (clone: 53–6.7; BioLegend), anti-CD11a-PE/Cy7 (clone: 2D7; BD Bioscience), anti–CD44-Alexa Fluor 700 (clone: IM7; BioLegend), anti-CD45-APC-eFluor780 (clone:30-F11;Biolegend), anti-CD62L-eVolve 605 (clone: MEL-14; eBioscience), anti-CD127-PE & PE/Cy7 (clone: A7R34; BioLegend), anti-KLRG1-FITC & BV510 (clone: 2F1/KLRG1; BioLegend), anti-CD103-APC (clone: 2E7; BioLegend), anti-CD69-FITC (clone: H1.2F3; BioLegend) and anti-IFNγ-APC (clone: XMG1.2; BioLegend), anti-Eomes-PE & PE/Cy7 (clone: Dan11mag; eBioscience).
Zheng X., Oduro J.D., Boehme J.D., Borkner L., Ebensen T., Heise U., Gereke M., Pils M.C., Krmpotic A., Guzmán C.A., Bruder D, & Čičin-Šain L. (2019). Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge. PLoS Pathogens, 15(9), e1008036.
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