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Clone ucht1

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The Clone UCHT1 is a monoclonal antibody that recognizes the CD3 epsilon subunit of the T cell receptor complex. It is commonly used in flow cytometry and other immunological applications to identify and quantify T cells.

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Lab products found in correlation

Clone cd28, by BioLegend (6 mentions) Cd8 microbead, by Miltenyi Biotec (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Fixation permeabilization concentrate, by Thermo Fisher Scientific (1 mentions) Anti human cd8, by BD (1 mentions) Clone mq1 17h12, by BioLegend (1 mentions) Clone rpa t8, by BD (1 mentions) Clone uchl1, by BD (1 mentions) Celltrace violet, by Thermo Fisher Scientific (1 mentions) Facsymphony a1, by BD (1 mentions) Fbs aim 5 medium, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Click it plus edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions) Collagenase type 1, by Merck Group (1 mentions) Clone rpa t4, by BD (1 mentions) Il 27, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions) Human cd4 t cell isolation kit, by STEMCELL (1 mentions) Il 12, by BioLegend (1 mentions) Facsdiva, by BD (1 mentions)

Spelling variants of this product

CD3 (clone UCHT1; (7 citations) Anti-CD3-FITC (clone UCHT1, (6 citations) Anti-humanCD3 (clone UCHT1, (4 citations) Anti-CD3 BV421 (clone UCHT1) (3 citations) Anti-CD3-APC-Cy7 clone UCHT1 (2 citations) Mouse anti-human CD3 (clone UCHT1 (2 citations) Pacific Blue-conjugated anti-human CD3 (clone UCHT1; (2 citations) Soluble anti-CD3 (clone UCHT1, (2 citations) Anti-CD3ε-APC (clone UCHT1, (2 citations) Anti-CD3 (clone OKT3 and clone UCHT1); anti-CD4 (clone RPA-T4); anti-CD11c (clone 3.9); anti-CD14 (clone M5E2); anti-CD15 (clone W6D3); anti-CD16 (clone 3G8); anti-CD19 (clone HIB19); anti-CD20 (clone 2H7); (2 citations)

15 protocols using clone ucht1

1

Multicolor Flow Cytometry of T Cells

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An aliquot of the T cell-enriched samples from each donor was stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend, San Diego, CA, USA) for 30 minutes at room temperature to validate the isolation of T cells. The aliquots were also stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend), anti-human CD8 (cat No. 560662, clone RPA-T8, BD Biosciences, Franklin Lakes, NJ, USA), and anti-human CD45RO (cat No. 562299, clone UCHL1, BD Biosciences). The cells were then treated with fixation/permeabilization concentrate (cat No. 00-5123-43, Invitrogen, Waltham, MA, USA) to fix and permeabilize cells for 1 h at 4°C, and intracellular staining was conducted according to the manufacturer’s recommendations. The cells were stained with anti-human interferon γ (IFN-γ) (cat No. 56-7319-41, clone 4S.B3, Invitrogen), anti-human tumor necrosis factor α (TNF-α) (cat No. 25-7349-41, clone MAb11, Invitrogen), and anti-human IL-2 (cat No. 500307, clone MQ1-17H12, BioLegend). Sample data were acquired using the LSR Fortessa X20 and were analyzed using FlowJo software.
Lee J.H., Lee B.H., Jeong S., Joh C.S., Nam H.J., Choi H.S., Sserwadda H., Oh J.W., Park C.G., Jin S.P, & Kim H.J. (2023). Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/ionomycin- and αCD3/αCD28-activated primary human T cells. Genomics & Informatics, 21(2), e18.
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2

T Cell Proliferation Assay with OX40 Activation

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T cells (1 × 106 cells/mL in DPBS) were labeled with 5 μM of CellTrace Violet (Life Technologies) and incubated at 37 °C for 20 min. Medium containing 2% FBS was added (five times the original volume of the staining medium) and centrifuged once to remove free dye. The cell pellet was resuspended in 10% FBS-AIM-V medium (Life Technologies) at 1 × 106 cells/mL, and 100 μL aliquots of the cells were added into wells precoated with 100 μL of anti-CD3 antibody (0.5 μg/mL, clone UCHT-1, Biolegend) and fusion antibodies to OX40 (0.25, 0.5, 1.0 and 2.0 μg/mL); soluble anti-CD28 (1 μg/mL; clone CD28.2, Biolegend) was added to the wells containing T cells. T cells in culture medium alone served as the non-stimulated cell control. The cells were then kept at 37 °C in a CO2 incubator for 5 days. They were then harvested and the reduction of CellTrace Violet (indicating cell proliferation) was assessed via flow cytometry (BD FACSymphony A1, BD biosciences).
Mahasongkram K., Glab-ampai K., Kaewchim K., Saenlom T., Chulanetra M., Sookrung N., Nathalang O, & Chaicumpa W. (2023). Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer. Vaccines, 11(12), 1826.
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3

Macrophage Engulfment of Opsonized and Necrotic Cells

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Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25μg/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2×106 IgGJ were injected IP.
McCubbrey A.L., McManus S.A., McClendon J.D., Thomas S.M., Chatwin H.B., Reisz J.A., D’Alessandro A., Mould K.J., Bratton D.L., Henson P.M, & Janssen W.J. (2022). Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells. Cell reports, 38(2), 110222.
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4

T-cell Proliferation Assay with HLA-E Peptides

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T-cell proliferation in response to HLA-E-binding peptides was assessed with the Click-iT Plus EdU flow cytometry assay kit (Invitrogen) following the manufacturer’s instructions. Briefly, 0.2×106 PBMCs/well were cultured for 10 days in 96-well round bottom culture plates in the presence of 10 μg/ml peptides or control stimuli. Each condition included 6 replicates and medium (IMDM 10% human serum with 10 ng/ml IL-7) without stimuli was used as negative control. As positive control, we included 2 μg/ml of phytohemagglutinin (PHA), 5 μg/ml of purified protein derivative (PPD), and 5 μg/ml of fusion early secretory antigenic target-10kDa culture filtrate protein (ESAT6/CFP10). On day 5 PBMCs were restimulated with peptide or control proteins and 100 U/ml IL-2 was added to the medium. 24 hours before harvesting for FACS staining and Click reaction, EdU was added to a final concentration of 25 μM. Once harvested, cells were washed with PBS and cell-surface antigens were antibody-stained before fixation/permeabilization and click reaction were subsequently performed. Fluorochrome-conjugated antibodies against human CD3 (clone UCHT1, Biolegend) and CD8 (clone SK1, Biolegend) samples were acquired on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software v10.7.1 (BD Biosciences).
Ruibal P., Franken K.L., van Meijgaarden K.E., van Wolfswinkel M., Derksen I., Scheeren F.A., Janssen G.M., van Veelen P.A., Sarfas C., White A.D., Sharpe S.A., Palmieri F., Petrone L., Goletti D., Abeel T., Ottenhoff T.H, & Joosten S.A. (2022). Identification of novel HLA-E-binding Mtb-derived epitopes through improved prediction models. Journal of immunology (Baltimore, Md. : 1950), 209(8), 1555-1565.
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5

Adipose Tissue Immune Cell Isolation

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AT from dermolipectomies was digested using type I collagenase (Sigma-Aldrich). Whole AT was sequentially digested with 1:1 dispase (2.4 U/mL in phosphate-buffered saline (PBS), Gibco, 30 min at 37 °C with shaking) and type I collagenase (250 U/mL in PBS 2% bovine serum albumin (BSA), Sigma-Aldrich, 30 min at 37 °C with shaking). After digestion, the cell suspension was filtered through a 250-µm strainer and centrifuged. The erythrocyte lysis step was performed followed by successive filtrations through 100, 70, and 40 -µm strainers. The viable recovered cells were counted and further analyzed by flow cytometry. Immune cells were enriched using a CD45 isolation kit (Stem Cells Technologies) following the manufacturer’s protocol. Anti-human FITC-CD4 dilution (1/10), BD Pharmingen, Ref 555346, CLONE RPA-T4 (RUO); PerCP-CD8 dilution (1/10), BD, Ref 345774, CLONE SK1 (CE/IVD); Pe-Cy7-CD56 dilution (1/20); BD Pharmingen, Ref 345774, CLONE B159 (RUO); APC-Cy7-CD19 dilution (1/20); BD Pharmingen, Ref 557791, CLONE SJ25C1 (RUO); APC-CD25 dilution (1/20), BD, ref 340907, CLONE 2A3 (CE/IVD); V450-CD3 diution (1/20); BD Horizon, ref 560365, CLONE UCHT1 (RUO); BV510-CD45 dilution (1/20), BIOLEGEND, ref 304036, CLONE HI30.Flow cytometry was carried out with LSRII BD Fortessa. Cells are expressed as number/g adipose tissue.
Zamora A., Nougué M., Verdu L., Balzan E., Draia-Nicolau T., Benuzzi E., Pujol F., Baillif V., Lacazette E., Morfoisse F., Galitzky J., Bouloumié A., Dubourdeau M., Chaput B., Fazilleau N., Malloizel-Delaunay J., Bura-Rivière A., Prats A.C, & Garmy-Susini B. (2024). 15-Lipoxygenase promotes resolution of inflammation in lymphedema by controlling Treg cell function through IFN-β. Nature Communications, 15, 221.
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6

Macrophage Engulfment of Opsonized and Necrotic Cells

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Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25μg/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2×106 IgGJ were injected IP.
McCubbrey A.L., McManus S.A., McClendon J.D., Thomas S.M., Chatwin H.B., Reisz J.A., D’Alessandro A., Mould K.J., Bratton D.L., Henson P.M, & Janssen W.J. (2022). Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells. Cell reports, 38(2), 110222.
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7

Isolation and Activation of CD4+ T Cells

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PBMCs were isolated from healthy volunteers. CD4+ T cells were isolated using a human CD4+ T cell isolation kit (STEMCELL Technologies), according to manufacturer’s guidelines. CD4+ T cells (numbering 1 × 106) were then cultured with soluble αCD28 (5 μg/mL, clone CD28.2, BioLegend) and plate bound αCD3ε (wells coated with 1 μg/mL for 4 hours at 37°C, 5% CO2, clone UCHT1, BioLegend), supplemented with 50 IU/mL IL-2 (Miltenyi Biotech), 10 ng/mL IL-12 (BioLegend), 100 ng/mL IL-27 (Thermo Fisher Scientific) cell polarizing cytokines in a 48-well plate (for a final volume of 500 μL). After 3 days, cell culture supernatants were collected and stored at –20°C, and cells were assessed by flow cytometry.
Edwards C.L., Ng S.S., de Labastida Rivera F., Corvino D., Engel J.A., Montes de Oca M., Bukali L., Frame T.C., Bunn P.T., Chauhan S.B., Singh S.S., Wang Y., Na J., Amante F.H., Loughland J.R., Soon M.S., Waddell N., Mukhopadhay P., Koufariotis L.T., Johnston R.L., Lee J.S., Kuns R., Zhang P., Boyle M.J., Hill G.R., McCarthy J.S., Kumar R, & Engwerda C.R. (2023). IL-10-producing Th1 cells possess a distinct molecular signature in malaria. The Journal of Clinical Investigation, 133(1), e153733.
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8

Characterizing Human Cell Engraftment in Humanized Mice

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For the analysis of human cell engraftment in humanized mice, recipient splenocytes and bone marrow cells were stained with Zombie live/dead stain (cat.no. 77184, Biolegend), anti-human CD45 (clone HI30, cat.no. 304008, 1:20 dilution, Biolegend with IgG1, PE, clone MOPC-21, cat.no. 400140 1:20 dilution, Biolegend), anti-human CD3 (clone UCHT1, cat.no. 300415, 1:20 dilution, Biolegend with IgG1, AF488, clone MOPC-21, cat.no. 400129 1:20 dilution, Biolegend), anti-human CD19 (clone HIB19, cat.no. 302228, 1:20 dilution, Biolegend with IgG1, PerCP, clone MOPC-21, cat.no. 400148 1:20 dilution, Biolegend) and anti-human CD33 (clone P67.6, cat.no. 366612, 1:20 dilution, Biolegend with IgG1, BV605, clone MOPC-21, cat.no. 400162 1:20 dilution, Biolegend). Cells were measured by flow cytometry (BD Aria and FACSDiva version 9.0 or CytExpert version 2.4 software) and gated for live cells, CD45 + cells and then followed by CD3 + cells, CD19 + or CD33 + cells. Results were expressed as percentage of positive population compared to control samples in FlowJo (BD).
Hu X., Manner K., DeJesus R., White K., Gattis C., Ngo P., Bandoro C., Tham E., Chu E.Y., Young C., Wells F., Basco R., Friera A., Kangeyan D., Beauchesne P., Dowdle W.E., Deuse T., Fry T.J., Foster A.E, & Schrepfer S. (2023). Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice. Nature Communications, 14, 2020.
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9

Comparative Immunosuppression of T-cell Subsets

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To compare the immunosuppressive functions of CD73+Vδ1 T, CD73-Vδ1 T and CD4+CD25+ T cells, the corresponding cells were sorted from BC specimens and then co-cultured with allogeneic CD4+ or CD8+ T cells from peripheral blood in the presence of IL-2 (40 U/ml, Peprotech), anti-CD3 antibody (10 μg/ml, clone UCHT1, BioLegend) and anti-CD28 antibody (10 μg/ml, clone CD28.2, BioLegend). The IFN-γ (BioLegend), Perforin (Abcam) and Granzyme B (BioLegend) levels were detected with corresponding ELISA kits.
Ni C., Fang Q.Q., Chen W.Z., Jiang J.X., Jiang Z., Ye J., Zhang T., Yang L., Meng F.B., Xia W.J., Zhong M, & Huang J. (2020). Breast cancer-derived exosomes transmit lncRNA SNHG16 to induce CD73+γδ1 Treg cells. Signal Transduction and Targeted Therapy, 5, 41.
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10

Naïve CD8+ T Cell Activation and Differentiation

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Peripheral blood was procured from healthy volunteers. Mononuclear cells were enriched by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare) in SepMate-50 tubes (Stem Cell Technologies). CD8+ T cells were isolated from PBMCs using CD8 microbeads (Miltenyi) according to manufacturer protocol. Cells were further stained with antibodies against CD8, CD62L, CCR7 and CD45RA. Naïve cells CD8+CD62LhiCCR7+CD45RA+ cells were sorted by BD FacsAria (BD Biosciences). Sorted CD8+ T cells were cultured for 9 days in RPMI supplemented with 10% (vol/vol) autologous heat-inactivated serum, 1 mM sodium pyruvate, 1X nonessential amino acids, 2mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. Naïve CD8+cells were stimulated with plate-bound anti-CD3 (Biolegend, clone UCHT1, 1 μg/ml) and anti-CD28 (Biolegend, clone CD28.2, 1μg/ml) in the presence of 10nM dexamethasone (Sigma) or vehicle control for 3 days. Cells were then rested in the presence of 100U/ml IL2 (R&D Systems) for 3 days. Next, the cells were restimulated with plate-bound anti-CD3 (1 μg/ml)and anti-CD28 (1μg/ml) in the presence of either 10nM dexamethasone (Sigma) or vehicle control for 3 days.
Acharya N., Madi A., Zhang H., Klapholz M., Escobar G., Dulberg S., Christian E., Ferreira M., Dixon K.O., Fell G., Tooley K., Mangani D., Xia J., Singer M., Bosenberg M., Neuberg D., Rozenblatt-Rosen O., Regev A., Kuchroo V.K, & Anderson A.C. (2020). Endogenous glucocorticoid signaling regulates effector differentiation and development of dysfunction in CD8+ T cells in the tumor microenvironment. Immunity, 53(3), 658-671.e6.
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