Anti cd3 apc efluro 780

Manufactured by Thermo Fisher Scientific

The Anti-CD3-APC-eFluro 780 is a fluorescently-labeled antibody that binds to the CD3 antigen on the surface of T cells. It can be used for the detection and enumeration of T cells in flow cytometry applications.

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Lab products found in correlation

Facsaria 3, by BD (6 mentions) Rnasin ribonuclease inhibitor, by Promega (6 mentions) Zombie nir, by BioLegend (6 mentions) Anti cd20 pecy7, by BD (6 mentions) Pan b cell isolation kit, by Miltenyi Biotec (5 mentions) Anti cd8 apc efluro 780, by Thermo Fisher Scientific (3 mentions) Anti cd16 apc efluro 780, by Thermo Fisher Scientific (3 mentions) Anti cd14 apc efluro 780, by Thermo Fisher Scientific (3 mentions) Anti cd8 apc efluor780, by Thermo Fisher Scientific (3 mentions) Anti cd14 apc efluor 780, by Thermo Fisher Scientific (3 mentions) Anti cd16 apc efluor 780, by Thermo Fisher Scientific (3 mentions) Facsdiva, by BD (2 mentions) Cd19 microbeads, by Miltenyi Biotec (1 mentions) Facsdiva software, by BD (1 mentions) Zombienir, by BD (1 mentions)

Spelling variants of this product

Anti-CD3 (512 citations) CD3-APC (57 citations) Anti-CD3-APC (40 citations)

6 protocols using anti cd3 apc efluro 780

Isolation and Characterization of SARS-CoV-2 Specific B Cells

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PBMCs were enriched for B cells by negative selection using a pan B cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1 X Phosphate-buffered Saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti-human antibodies: anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluro 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluro 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluro 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105), and fluorophore-labeled RBD and Ovalbumin for 30 minutes on ice^{27 (link)}. Single CD3⁻CD8⁻CD16⁻CD20⁺Ova⁻RBD-PE⁺RBD-AF647⁺ B cells were sorted into individual wells of a 96-well plates containing 4 μl of lysis buffer (0.5 X PBS, 10mM DTT, 3000 units/mL RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III (Becton Dickinson). The sorted cells were frozen on dry ice, and then stored at −80°C or immediately used for subsequent RNA reverse transcription. Although cells were not stained for IgG expression, they are memory B cells based on the fact that they are CD20+ (a marker absent in plasmablasts) and they express IgG (since antibodies were amplified from these cells using IgG-specific primers).

Robbiani D.F., Gaebler C., Muecksch F., Lorenzi J.C., Wang Z., Cho A., Agudelo M., Barnes C.O., Gazumyan A., Finkin S., Hagglof T., Oliveira T.Y., Viant C., Hurley A., Hoffmann H.H., Millard K.G., Kost R.G., Cipolla M., Gordon K., Bianchini F., Chen S.T., Ramos V., Patel R., Dizon J., Shimeliovich I., Mendoza P., Hartweger H., Nogueira L., Pack M., Horowitz J., Schmidt F., Weisblum Y., Michailidis E., Ashbrook A.W., Waltari E., Pak J.E., Huey-Tubman K.E., Koranda N., Hoffman P.R., West AP J.r., Rice C.M., Hatziioannou T., Bjorkman P.J., Bieniasz P.D., Caskey M, & Nussenzweig M.C. (2020). Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals. bioRxiv.

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Isolation and Sorting of EDIII-Specific Memory B Cells

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PBMCs from sample 111 were enriched for B cells via positive selection using CD19 microbeads (Miltenyi Biotec; 130–050-301). PBMCs from all other donors were enriched for B cells by negative selection (Miltenyi Biotec; 130–101-638). All selection protocols were performed according to the manufacturer’s instructions. Enriched B cells were incubated for 30 min on ice in FACS buffer (1× PBS, 2% calf serum, 1 mM EDTA) with fluorophore-labeled EDIII and ovalbumin, and in the presence of anti-human antibodies anti-CD3-APC-eFluro 780 (Invitrogen; 47–0037-41), anti-CD8-APC-eFluro 780 (Invitrogen; 47–0086-42), anti-CD14-APC-eFluro 780 (Invitrogen; 47–0149-42), anti-CD16-APC-eFluro 780 (Invitrogen; 47–0168-41), anti-CD20-PECy7 (BD Biosciences; 335793), and Zombie NIR (BioLegend; 423105). Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR⁻CD20⁺Ova⁻EDIII-PE⁺EDIII-AF647⁺ B cells were sorted using a FACS Aria III (Becton Dickinson) into individual wells of 96-well plates. Each well contained 4 µl of a lysis buffer comprising 0.5× PBS, 10 mM DTT, and 3,000 U/ml RNasin Ribonuclease Inhibitors (Promega; N2615). Sorted cells were snap-frozen on dry ice and then stored at −80°C. Antibody sequences are derived from memory B cells because they originate from small CD20⁺ cells, and the antibody genes were PCR amplified using IgG-specific primers.

Agudelo M., Palus M., Keeffe J.R., Bianchini F., Svoboda P., Salát J., Peace A., Gazumyan A., Cipolla M., Kapoor T., Guidetti F., Yao K.H., Elsterová J., Teislerová D., Chrdle A., Hönig V., Oliveira T., West AP J.r., Lee Y.E., Rice C.M., MacDonald M.R., Bjorkman P.J., Růžek D., Robbiani D.F, & Nussenzweig M.C. (2021). Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease. The Journal of Experimental Medicine, 218(5), e20210236.

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Single-cell sorting for SARS-CoV-2 antibody discovery

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Single cell sorting by flow cytometry was performed as described (Robbiani et al., 2020 (link)). Briefly, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3−CD8−CD14−CD16−CD20+Ova−Gamma NTD-PE⁺Wuhan-Hu-1 NTD-AF647⁺ B cells were sorted into individual wells of 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 FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Wang Z., Muecksch F., Cho A., Gaebler C., Hoffmann H.H., Ramos V., Zong S., Cipolla M., Johnson B., Schmidt F., DaSilva J., Bednarski E., Tanfous T.B., Raspe R., Yao K., Lee Y.E., Chen T., Turroja M., Milard K.G., Dizon J., Kaczynska A., Gazumyan A., Oliveira T.Y., Rice C.M., Caskey M., Bieniasz P.D., Hatziioannou T., Barnes C.O, & Nussenzweig M.C. (2022). Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins. bioRxiv.

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Isolation and Characterization of SARS-CoV-2 RBD-Specific B Cells

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As previously described (Robbiani et al., 2020 (link)), PBMCs were enriched for B cells via negative selection using a pan–B cell isolation kit (130-101-638; Miltenyi Biotec) according to the manufacturer’s protocol. Enriched B cells were incubated with fluorophore-labeled RBD and ovalbumin, and in the presence of antihuman antibodies anti-CD3-APC-eFluro 780 (47-0037-41; Invitrogen), anti-CD8-APC-eFluro 780 (47-0086-42; Invitrogen), anti-CD14-APC-eFluro 780 (47-0149-42; Invitrogen), anti-CD16-APC-eFluro 780 (47-0168-41; Invitrogen), anti-CD20-PECy7 (335793; BD Biosciences), and Zombie NIR (423105; BioLegend) in FACS buffer (1 × PBS, 2% calf serum, 1mM EDTA) for 30 min on ice. Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR^-CD20⁺Ova⁻RBD⁺RBD KEN⁺ were sorted using a FACS Aria III (Becton Dickinson) into individual wells of a 96-well plate, each containing 4 μl of lysis buffer comprising 0.5× PBS, 10 mM dithiothreitol, and 3,000 U/ml RNasin Ribonuclease Inhibitors (N2615; Promega). Sorted cells were frozen on dry ice and stored at −80°C until further processing.

Agudelo M., Muecksch F., Schaefer-Babajew D., Cho A., DaSilva J., Bednarski E., Ramos V., Oliveira T.Y., Cipolla M., Gazumyan A., Zong S., Rodrigues D.A., Lira G.S., Conde L., Aguiar R.S., Ferreira OC J.r., Tanuri A., Affonso K.C., Galliez R.M., Castineiras T.M., Echevarria-Lima J., Bozza M.T., Vale A.M., Bieniasz P.D., Hatziioannou T, & Nussenzweig M.C. (2022). Plasma and memory antibody responses to Gamma SARS-CoV-2 provide limited cross-protection to other variants. The Journal of Experimental Medicine, 219(9), e20220367.

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Single-cell sorting of SARS-CoV-2 specific B cells

Cited 1 time

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Single cell sorting by flow cytometry was performed as described (Robbiani et al., 2020 (link)). Briefly, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3−CD8−CD14−CD16−CD20+Ova−Gamma NTD-PE⁺Wuhan-Hu-1 NTD-AF647⁺ B cells were sorted into individual wells of 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 FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Wang Z., Muecksch F., Cho A., Gaebler C., Hoffmann H.H., Ramos V., Zong S., Cipolla M., Johnson B., Schmidt F., DaSilva J., Bednarski E., Ben Tanfous T., Raspe R., Yao K., Lee Y.E., Chen T., Turroja M., Milard K.G., Dizon J., Kaczynska A., Gazumyan A., Oliveira T.Y., Rice C.M., Caskey M., Bieniasz P.D., Hatziioannou T., Barnes C.O, & Nussenzweig M.C. (2022). Analysis of memory B cells identifies conserved neutralizing epitopes on the N-terminal domain of variant SARS-Cov-2 spike proteins. Immunity, 55(6), 998-1012.e8.

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Single-cell Sorting and Isolation of SARS-CoV-2 Reactive B Cells

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Single-cell sorting by flow cytometry was previously described^{1 (link)}. In brief, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130–101-638). The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20–PECy7 (BD Biosciences, 335793), anti-CD3–APC–eFluro 780 (Invitrogen, 47–0037-41), anti-CD8–APC–eFluor 780 (Invitrogen, 47–0086-42), anti-CD16–APC–eFluor 780 (Invitrogen, 47–0168-41), anti-CD14–APC–eFluor 780 (Invitrogen, 47–0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3⁻CD8⁻CD14⁻CD16⁻CD20⁺Ova⁻RBD– PE⁺RBD–AF647⁺ B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units per ml RNasin ribonuclease inhibitors (Promega, N2615)) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Gaebler C., Wang Z., Lorenzi J.C., Muecksch F., Finkin S., Tokuyama M., Cho A., Jankovic M., Schaefer-Babajew D., Oliveira T.Y., Cipolla M., Viant C., Barnes C.O., Bram Y., Breton G., Hägglöf T., Mendoza P., Hurley A., Turroja M., Gordon K., Millard K.G., Ramos V., Schmidt F., Weisblum Y., Jha D., Tankelevich M., Martinez-Delgado G., Yee J., Patel R., Dizon J., Unson-O’Brien C., Shimeliovich I., Robbiani D.F., Zhao Z., Gazumyan A., Schwartz R.E., Hatziioannou T., Bjorkman P.J., Mehandru S., Bieniasz P.D., Caskey M, & Nussenzweig M.C. (2021). Evolution of antibody immunity to SARS-CoV-2. Nature, 591(7851), 639-644.

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