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Anti cd3 af700

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Anti-CD3-AF700 is a fluorescently-labeled antibody that binds to the CD3 antigen, which is expressed on the surface of T cells. It can be used for the detection and analysis of T cells in various research and diagnostic applications.

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Lab products found in correlation

Anti cd4 fitc, by Thermo Fisher Scientific (2 mentions) Anti cd45 af700, by BD (2 mentions) Anti cd8a pe, by BD (2 mentions) Anti cd19 af647, by BD (2 mentions) Anti cd11c af488, by BD (2 mentions) Anti gr1 v450, by BD (2 mentions) Anti mhcii apc, by Thermo Fisher Scientific (2 mentions) Live dead stain, by Thermo Fisher Scientific (2 mentions) Anti siglecf pe, by BD (2 mentions) Anti cd45 ef450, by Thermo Fisher Scientific (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Anti ifn γ apc, by BD (2 mentions) Anti mip1β pe, by BD (2 mentions) Anti cd16 apc cy7, by Thermo Fisher Scientific (2 mentions) Anti cd56 pe cy7, by Thermo Fisher Scientific (2 mentions) Anti cd107a pe cy5, by BD (2 mentions) Fix perm kit, by Thermo Fisher Scientific (2 mentions) Brefeldin a, by Merck Group (2 mentions) Golgistop, by BD (2 mentions) Lsr 2 flow cytometer, by BD (2 mentions)

6 protocols using anti cd3 af700

1

Mouse Bone Marrow and Spleen Cell Isolation

Cited 2 times
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Bone marrow cells were collected from the femurs and tibiae of mice by flushing the opened bones with Iscove’s Modified Dulbecco’s medium (IMDM) as previously described (Dyer et al., 2008 (link)). Spleen cell suspensions in HBSS supplemented with 1% FBS and 10 mM HEPES were prepared as described (Dyer et al., 2011 (link)). Red blood cells were lysed with ACK lysing buffer (Lonza). Live/dead stain (Invitrogen) was added to the cells and non-specific antibody binding to Fc receptors was blocked with anti-mouse CD16/CD32 (BD Biosciences). For analysis of T and B cells, cell suspensions were incubated with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in phosphate-buffered saline with 0.1% bovine serum albumin (PBS/BSA) at 4°C for 30 min and washed with this buffer prior to analysis. For evaluation of myeloid cells, cell suspensions were incubated with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). At least 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.
Rice T.A., Brenner T.A., Percopo C.M., Ma M., Keicher J.D., Domachowske J.B, & Rosenberg H.F. (2016). Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection. Antiviral research, 132, 131-140.
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2

Characterization of Lung Cell Populations

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Lung tissue was harvested and single-cell suspensions were prepared as previously described (Garcia-Crespo et al., 2013 (link); Percopo et al., 2014a (link)). Live/Dead stain (Invitrogen) was added to the cells and Ab binding to Fc receptors was blocked with anti-mouse CD16/CD32. For analysis of T and B cells, lung suspensions were stained with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in PBS with 0.1% BSA at 4 °C for 30 min and washed with this buffer prior to analysis. For myeloid cell analysis, cells were stained with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). Natural killer cells were characterized by staining with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (BD), and anti-CD49/DX5-PE (BD). A minimum of 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.
Percopo C.M., Rice T.A., Brenner T.A., Dyer K.D., Luo J.L., Kanakabandi K., Sturdevant D.E., Porcella S.F., Domachowske J.B., Keicher J.D, & Rosenberg H.F. (2015). Immunobiotic Lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection. Antiviral research, 121, 109-119.
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3

Multiparametric Immune Profiling of PBMCs

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After PBMCs stimulation, cells were washed with PBS and incubated at 4°C for 30 minutes with Fixable Viability Dye eFluor 506 (eBioscience), and with the anti-CD3-AF700 (clone: UCHT1, eBioscience) and anti-CD8-eFluor450 (clone: RPA-T8, eBiocience) antibodies. Next, cells were fixed and permeabilized with Foxp3 Fixation/Permeabilization Buffer (eBioscience) and then incubated with the following conjugated antibodies: anti-IL-2- (clone: 5344.111, BD), anti-Granzyme B (clone: GB11, BD), anti-Perforin (clone: B-D48, Biolegend), anti-IFN-γ (clone: 4S.B3, Biolegend), anti-TNF-α (clone: MAb11, eBioscience) and anti-IL-10 (clone: JES3-9D3, eBioscience). Cells were acquired on an LSR Fortessa flow cytometer using the BD FACSDiva software v 8.0.1 (BD). At least 100,000 CD3+ events were recorded. Fluorescence minus one (FMO) control for the effector molecules was included to define positive thresholds. For fluorochrome spillover compensation, unstained and single-stained cells were used with each of the fluorochrome-labeled antibodies. Then, automatic compensation was performed on LSR Fortessa.
Sánchez-Martínez A., Acevedo-Sáenz L., Alzate-Ángel J.C., Álvarez C.M., Guzmán F., Roman T., Urcuqui-Inchima S., Cardona-Maya W.D, & Velilla P.A. (2022). Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia. Frontiers in Immunology, 13, 793982.
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4

NK Cell Degranulation Assay

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The capacity of serum to promote NK degranulation was performed as in Chung et al., (2014) and Sips et al. (2016) (link). Targets were prepared by pulsing CEM-NKr-CCR5 cells with gp120 YU-2 (ImmuneTech, 50 μg/ml) as previously described (Chung et al., 2014 ). After washing excess gp120, the pulsed cells were co-cultured with primary NK cells (prepared as by negative selection as above) at a ratio of 5:1 effectors: target, in the presence of diluted donor plasma (1:100 final dilution), anti-CD107a-PE-Cy5 (BD), brefeldin A (10 mg/ml, Sigma), and GolgiStop (BD) for 5 hours at 37°C. After incubation, cells were stained for surface markers with anti-CD16 APC-Cy7, anti-CD56 PE-Cy7, and anti-CD3 AF700, washed, followed by fixation and permeabilization with Fix & Perm kit (ThermoFisher). Intracellular staining was performed with anti–IFN-γ–APC (BD) and anti–MIP-1β–PE (BD). Cells were analyzed on a BD LSRII flow cytometer. NK cells were defined as CD3-negative, and CD16+ and/or CD56+.
Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.
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5

NK Cell Degranulation Assay

Cited 1 time
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The capacity of serum to promote NK degranulation was performed as in Chung et al., (2014) and Sips et al. (2016) (link). Targets were prepared by pulsing CEM-NKr-CCR5 cells with gp120 YU-2 (ImmuneTech, 50 μg/ml) as previously described (Chung et al., 2014 ). After washing excess gp120, the pulsed cells were co-cultured with primary NK cells (prepared as by negative selection as above) at a ratio of 5:1 effectors: target, in the presence of diluted donor plasma (1:100 final dilution), anti-CD107a-PE-Cy5 (BD), brefeldin A (10 mg/ml, Sigma), and GolgiStop (BD) for 5 hours at 37°C. After incubation, cells were stained for surface markers with anti-CD16 APC-Cy7, anti-CD56 PE-Cy7, and anti-CD3 AF700, washed, followed by fixation and permeabilization with Fix & Perm kit (ThermoFisher). Intracellular staining was performed with anti–IFN-γ–APC (BD) and anti–MIP-1β–PE (BD). Cells were analyzed on a BD LSRII flow cytometer. NK cells were defined as CD3-negative, and CD16+ and/or CD56+.
Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.
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6

Basophil Identification and Stimulation Protocol

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Whole blood was collected in a heparin and an EDTA tube. The EDTA tube was used for external CBC analysis. The heparin tube was kept at 18–25°C. Basophil identification was done using unstimulated blood (PBS) as well as blood stimulated with either Anti-IgE-FITC (Thermo Fisher, Waltham, MA) or fMLP (Sigma, St. Louis, MO). The samples were incubated for 20 min at 37°C followed by 10 min at 4°C (4 (link), 14 (link), 21 (link)–24 (link)). Each sample was stained with the following antibodies Anti-IgE-FITC (Clone Ige21), anti-CD193-PE (Clone 5E8), anti-CD123-PerCPCy5.5 (Clone 6H6), anti-CD203c-PECY7 (Clone NP4D6), anti-CRTH2-APC (Clone BM16), anti-CD3-AF700 (Clone UCHT1), anti-CD45-EF506 (Clone HI30) anti-FcɛRI-SB600 (Clone AER-37) (all Thermo Fisher, Waltham, MA) and anti-HLADR-Pacific blue (L243) (Biolegend, San Diego, CA) for 30 min at 4°C. Each antibody was titrated to obtain the best separation (25 (link)). The red blood cells were lysed using BD FACS lysis solutions (BD Bioscience, San Jose, CA) and resuspended in 400 μl PBS before acquisition.
Sonder S.U., Plassmeyer M., Loizou D, & Alpan O. (2023). Towards standardizing basophil identification by flow cytometry. Frontiers in Allergy, 4, 1133378.
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