Cd3 clone ucht1

Manufactured by BioLegend

Sourced in United States

CD3 (clone UCHT1) is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. The CD3 complex is essential for T cell activation and signal transduction.

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Anti-CD3 (clone UCHT1, (16 citations) Clone UCHT1 (15 citations) Anti-CD3-FITC (clone UCHT1, (6 citations) Anti-CD3-PerCP-cy5.5 (clone UCHT1; (4 citations) Anti-human CD3-AF700 antibody (clone UCHT1, (4 citations) CD3 PE-Cy7 (clone UCHT1, (4 citations) CD3-FITC clone UCHT1 (3 citations) Anti-CD3 BV421 (clone UCHT1) (3 citations) Mouse anti-human CD3 antibody (clone UCHT1, (3 citations) Anti-CD3-APC-Cy7 clone UCHT1 (2 citations)

7 protocols using cd3 clone ucht1

Immunophenotyping of Immune Cells post-HIIT

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Baseline and post-HIIT blood samples were collected in anticoagulant citrate dextrose solution (BD Diagnostics, Oakville, ON, Canada) and incubated in ACK lysis buffer for 5 minutes to remove red blood cells. Cells were blocked with CD16/CD32 antibody (eBioscience, San Diego, CA, USA), and surfaced stained with alexa fluor 700, phycoerthrin, and PerCP conjugated anti-mouse CD3 (clone 17A2; eBioscience), NK1.1 (clone PK136; BD Pharmingen) and CD27 (clone LG.7F9, eBioscience) antibodies, respectively. Human PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare) density-gradient centrifugation and surface stained using phycoerthrin and PerCP conjugated anti-human CD56 (clone B159; BD Pharmingen) and CD3 (clone UCHT1; BioLegend) antibodies, respectively. Stained cells fixed in 1% PFA were analyzed on a LSRII flow cytometer collecting 50,000 gated events and FlowJo flow cytometry analysis software.

Barra N.G., Fan I.Y., Gillen J.B., Chew M., Marcinko K., Steinberg G.R., Gibala M.J, & Ashkar A.A. (2017). High Intensity Interval Training Increases Natural Killer Cell Number and Function in Obese Breast Cancer-challenged Mice and Obese Women. Journal of Cancer Prevention, 22(4), 260-266.

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Humanized Mouse Model for Hematopoiesis

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All animal husbandry and experimental procedures were conducted in accordance with applicable federal and state guidelines and approved by the Animal Care and Use Committee of Columbia University. Mice were housed at the Columbia University animal facility (New York, NY) with a standard 12:12 h light-dark schedule and given water and regular rodent chow ad libitum.
Female immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice (Jackson Laboratory, Bar Harbor, ME), aged 6 to 8 weeks, were engrafted with commercially available human cord blood CD34⁺ cells (Cincinnati Children’s Hospital Medical Center; Cincinnati, OH). For the engraftment, the NSG mice were exposed to 2.0 Gy of γ-rays, followed by injection of 200,000 human CD34⁺ into the mouse tail vein.
Sixteen weeks later, human cell engraftment was assessed in the mouse blood by flow cytometry, using antibodies specific to human blood cells including CD45 clone Hl30 (leukocyte common antigen), CD3 clone UCHT1 (marker for T cells), CD19 clone 2H7 (marker for B cells), CD11b clone TCRF44 (marker for granulocytes, monocytes/macrophages) and mouse-specific CD45 antibody clone: 30-F11 (all from BioLegend^® Inc., San Diego, CA).

Pujol-Canadell M., Young E, & Smilenov L. (2019). Use of a Humanized Mouse Model System in the Validation of Human Radiation Biodosimetry Standards. Radiation research, 191(5), 439-446.

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Viability and Phenotypic Analysis of NK Cells

Cited 1 time

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Cell viability of fresh and cryopreserved NK cells is assessed by staining with the Zombie NIR dye (dilution 1:1000; Biolegend). Fresh and cryopreserved NK cells are phenotypically characterized as described in refs. ^{28 (link),29 (link)} by staining with directly conjugated mouse anti-human antibodies against CD3 (clone UCHT1; dilution 1:50; Biolegend), CD56 (clone HCD56; dilution 1:50; Biolegend), and CD16 (3G8; dilution 1:50; Biolegend). NK cells are defined as CD3− and CD56+ cells (Supplementary Fig. 7). A minimum of 10,000 cells are analyzed using a BD Canto II flow cytometer (BD Biosciences) and Flowjo Software (FLOWJO, LLC Data analysis software).

Mark C., Czerwinski T., Roessner S., Mainka A., Hörsch F., Heublein L., Winterl A., Sanokowski S., Richter S., Bauer N., Angelini T.E., Schuler G., Fabry B, & Voskens C.J. (2020). Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells. Nature Communications, 11, 5224.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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To be able to exclude dead cells from the analysis, PBMCs were incubated with the viability dye Zombie-NIR (1:1000 in PBS, Biolegend, San Diego, USA) for 30 min in the dark. After washing in PBS, cells were stained with antibodies against CD3 (clone UCHT1; 1:50) and CD8 (clone RPA-T8; 1:500; both from Biolegend, San Diego, USA) in PBS/2% bovine serum albumin (BSA) for 20 min at 4 °C in the dark. Then, cells were washed in PBS/2% BSA and permeabilized with Fixation and Permeabilization buffer (BD Pharmingen, Heidelberg, Germany) for 20 min at 4 °C in the dark. The cells were washed twice in Perm/Wash buffer (BD Pharmingen, Heidelberg, Germany) and stained intracellularly with antibodies against CD4 (clone RPA-T4; 1:20), IFNγ (clone 4S.B3; 1:100), IL-17 (clone BL168; 1:20), IL-22 (clone 2G12A41; 1:20) and TNF (clone MAb11; 1:100; all from Biolegend, San Diego, USA) in Perm/Wash buffer for 30 min at 4 °C in the dark. Afterwards, the cells were washed again in Perm/Wash buffer and resuspended in PBS. Analyses were performed with a BD LSR II flow cytometer (BD Biosciences, San José, USA) and FlowJo single cell analysis software (FlowJo LLC, Ashland, USA).

Rauch J., Jochum J., Eisermann P., Gisbrecht J., Völker K., Hunstig F., Mehlhoop U., Muntau B, & Tappe D. (2022). Inflammatory cytokine profile and T cell responses in African tick bite fever patients. Medical Microbiology and Immunology, 211(2-3), 143-152.

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TCR-α/β Expression Profiling in Jurkat Cells

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TCR

α β

- KO Jurkat E6.1 cells (a kind gift of Edward Jenkins) were transduced with 1G4 or A6 lentivirus, and TCR expression was measured by staining for CD3 (clone: UCHT1; Biolegend) and TCR

α β

(clone IP26; Biolegend).

Pettmann J., Huhn A., Abu Shah E., Kutuzov M.A., Wilson D.B., Dustin M.L., Davis S.J., van der Merwe P.A, & Dushek O. (2021). The discriminatory power of the T cell receptor. eLife, 10, e67092.

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Comprehensive Immune Cell Profiling

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Cell staining of whole blood was performed for 25 min on ice in the dark in staining buffer composed of PBS, 0.5% BSA, and 0.05% sodium azide. Red blood cells were lysed by addition of 1× BD Pharm Lyse Buffer (BD Biosciences) and incubation in the dark at room temperature for 15 min. Following one wash, cells were fixed in 2% paraformaldehyde for 15 min at room temperature, washed again, and resuspended in staining buffer. A minimum of 300,000 total events were collected on a FACS Calibur using Cell Quest and analyzed with FlowJo software (TreeStar). Anti-human CD20 (clone 2H7, catalog #302304), CD19 (clone HIB19, catalog #302234), CD38 (clone HB7, catalog #356608), CD3 (clone UCHT1, catalog #300426), CD4 (clone RPA-T3, catalog #300506), CD25 (clone BC96, catalog #302632), CD127 (clone A019D5, catalog #351325), CCR4 (clone TG6, catalog #335405), CXCR5 (clone TG2, catalog #335001), CD45RO (clone UCHL1, catalog #304218), and PD-1 (clone EH12.2.H7, catalog #329907) antibodies were purchased from BioLegend, anti-human CD27 (clone O323, catalog #12-0279-42) from eBioscience, and anti-human ICOS antibody (clone DX29, catalog #557802) from BD Biosciences.

Panwar B., Schmiedel B.J., Liang S., White B., Rodriguez E., Kalunian K., McKnight A.J., Soloff R., Seumois G., Vijayanand P, & Ay F. (2021). Multi–cell type gene coexpression network analysis reveals coordinated interferon response and cross–cell type correlations in systemic lupus erythematosus. Genome Research, 31(4), 659-676.

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Transduction and TCR Expression in Jurkat Cells

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TCRαβ-KO Jurkat E6.1 cells (a kind gift of Edward Jenkins) were transduced with 1G4 or A6 lentivirus and TCR expression was measured by staining for CD3 (clone: UCHT1; Biolegend) and TCRαβ (clone IP26; Biolegend).

Pettmann J., Abu‐Shah E., Kutuzov M.A., Wilson D.B., Dustin M.L., Davis S.J., Merwe P.A.v.d., & Dushek O. (2020). T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands.

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