Anti cd3 af700 clonehit3a

Manufactured by BioLegend

Anti-CD3-AF700 (clone HIT3a) is a fluorescence-labeled monoclonal antibody that specifically binds to the CD3 antigen expressed on T cells. It is used for the identification and enumeration of T cells by flow cytometry.

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Lab products found in correlation

Bv510 streptavidin, by BioLegend (2 mentions) Anti cd8 fitc clone sk1, by BioLegend (2 mentions) Anti granzymeb pacific blue, by BioLegend (2 mentions) Anti cd62l pe cy5 clone dreg 56, by BioLegend (2 mentions) Anti cd45ra bv605, by BioLegend (2 mentions) Anti granzyme a percp cy5, by BioLegend (2 mentions) Bv650 streptavidin, by R&D Systems (2 mentions) Anti granzyme k pe, by BioLegend (2 mentions) Anti granzyme m efluor660, by Thermo Fisher Scientific (2 mentions) Annexin 5 pe, by BD (1 mentions) Anti cd4 fitc clone rpa t4, by BioLegend (1 mentions) Facsdiva software, by BD (1 mentions) Lsrfortessa, by BD (1 mentions) Anti cd8 percp cy5, by Thermo Fisher Scientific (1 mentions) Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (1 mentions) Anti cd19 apc cy7, by Thermo Fisher Scientific (1 mentions) Ki67 bv421 clone b56, by BD (1 mentions)

Spelling variants of this product

Anti-CD3 (468 citations) CD3-AF700 (25 citations) Anti-CD3-AF700 (15 citations)

4 protocols using anti cd3 af700 clonehit3a

Assessing CD8+ T Cell Cytolytic Potential

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Rested ex vivo CD8⁺ T cells were assessed for CMV and
HIV specificity using HLA-matched tetramers. Tetramers were made via
biotinylated monomer conjugation to BV510 streptavidin (BioLegend). Monomers
were obtained from Dr. Soren Buus (University of Copenhagen). The A02-NV9
tetramer was used to stain for CMV-specificity while pools of A02-SL9, B27-KK10,
B53-QW9, B53-YY9, B57-IW9(p24), B57-IW9(RT), B57-KF11, B57-QW9, and B57-TW10
tetramers were used to stain for HIV-specificity. Subsequent surface stains
included anti-CD62L-PE-Cy5 (clone DREG-56; BioLegend), anti-CD3-AF700 (clone
HIT3a; BioLegend), anti-CD8-FITC (clone SK1; BioLegend), anti-CD45RA-BV605
(clone HI100; BioLegend), and LIVE/DEAD Blue. Following permeabilization, the
cells were intracellular stained using anti-perforin PE/Cy7 (clone B-D48;
BioLegend), anti-granzyme A-PerCp/Cy5.5 (clone CB9; BioLegend), anti-granzyme
B-Pacific Blue (clone GB11; BioLegend), anti-granzyme H (biotinylated and
secondary stained with BV650 streptavidin) (polyclonal antibody, R&D),
anti-granzyme K-PE (clone GM26E7; BioLegend), and anti-granzyme M-eFluor660
(clone 4B2G4; eBioscience). The cells were analyzed by flow cytometry.
Naïve CD8⁺ T cells within the mixed population of
CD8⁺ T cells were used as internal gating controls for
perforin and each granzyme as these cells do not express these effector
molecules.

Clayton K.L., Collins D.R., Lengieza J., Ghebremichael M., Dotiwala F., Lieberman J, & Walker B.D. (2018). Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation. Nature immunology, 19(5), 475-486.

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CD4 T Cell Viability Assay

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CD4 T cell clones were seeded in a 96-well V bottom plate and subjected to standard or optimized multimer staining procedures and put in culture in a round bottom 96-well plate in R8 media containing 100 IU/mL of hrIL-2. At time points 6 hour, 24 hours and 48 hours, the cells were collected and stained for viability markers. Cells were washed with PBS and resuspended in 25 µL of PBS containing anti-CD3 AF700 (clone HIT3a, Biolegend), anti-CD4 FITC (clone RPA-T4, Biolegend) and LIVE/DEAD fixable dead cell stain (Vivid, Invitrogen) diluted 1:800 in PBS. The cells were incubated at RT for 30 min and washed with PBS. They were resuspended in 25 µL of AnnexinV buffer 1X with AnnexinV-PE (Becton Dickinson) and incubated for 20 min at 4°C. Cells were then washed and resuspended in AnnexinV buffer 1X and analyzed with the CytoFLEX S Flow Cytometry Analyzer (Becton Dickinson).

Rockinger G.A., Guillaume P., Cachot A., Saillard M., Speiser D.E., Coukos G., Harari A., Romero P.J., Schmidt J, & Jandus C. (2020). Optimized combinatorial pMHC class II multimer labeling for precision immune monitoring of tumor-specific CD4 T cells in patients. Journal for Immunotherapy of Cancer, 8(1), e000435.

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Cytotoxic T Cell Assay for EBV-Associated Cancers

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EBV-associated cancer cells were plated at a density of 1×10⁵ cells/well. After 24 hours, T cells were added at an effector-to-target ratio of 50:1 and the culture was incubated for 24 hours at 37°C and 6.5% CO₂. To assess the impact of T cells on cancer cells, the cultured cells were then incubated at 4°C with the following antibodies: human anti-CD45-V500 (clone HI30, BD Biosciences), anti-CD3-AF700 (clone HIT3a, BioLegend), anti-CD56-BV421, anti-CD8-PerCP-Cy5.5, anti-CD19-APC-Cy7, anti-perforin-PE (clone dG9, eBioscience), anti-granzyme K-FITC (clone G3H69, BD Biosciences), anti-granzyme B-BV711 (clone GB11, BD Biosciences), Ki67-BV421 (clone B56, BD Biosciences), anti-active caspase-3-BV605 (clone C92-605, BD Biosciences) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific, MA). Flow cytometry was performed using a BD LSRFortessa with FACSDiva software and postacquisition analysis was performed using FlowJo software.

Sinha D., Srihari S., Beckett K., Le Texier L., Solomon M., Panikkar A., Ambalathingal G.R., Lekieffre L., Crooks P., Rehan S., Neller M.A., Smith C, & Khanna R. (2021). 'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies. Journal for Immunotherapy of Cancer, 9(2), e001608.

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Assessing CD8+ T Cell Cytolytic Potential

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