Anti cd3 okt3

Manufactured by BioLegend

Sourced in United States

Anti-CD3 (OKT3) is a monoclonal antibody that specifically binds to the CD3 complex on the surface of T cells. The CD3 complex is involved in the activation and signal transduction of T cells.

Automatically generated - may contain errors

Lab products found in correlation

Anti cd4 okt4, by BioLegend (5 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (5 mentions) Retronectin, by Takara Bio (4 mentions) Interleukin 2 (il 2), by BioLegend (3 mentions) Human ab serum, by Merck Group (3 mentions) Facs fortessa, by BD (2 mentions) Live dead near ir dye, by Thermo Fisher Scientific (2 mentions) Anti cd69 fn50, by BioLegend (2 mentions) Anti tcr β ip26, by BioLegend (2 mentions) Anti cd137 4b4 1, by BD (2 mentions) Fc blocking solution, by BioLegend (2 mentions) Anti cd8 sk1, by BioLegend (2 mentions) Anti cd45 h130, by BioLegend (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Anti cd56 5.1h11, by BioLegend (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Lsrfortessa, by BD (2 mentions) Anti cd56 hcd56, by BioLegend (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Anti-CD3 (468 citations) Anti-CD3 antibody (clone OKT3, (11 citations) Anti-human CD3 (OKT3, (5 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) BV785-conjugated anti-CD3 (clone OKT3; (2 citations) Plate-bound anti-CD3 (OKT3 - (2 citations) Anti-CD3 (OKT3) and anti-CD28 (CD28.2) (2 citations) Plate-bound anti-CD3 antibody (clone OKT3 – (2 citations) Soluble anti-CD3 (clone OKT3 (2 citations) APC anti-CD3 (clone OKT3, (2 citations)

41 protocols using anti cd3 okt3

Isolation and Activation of Human PBMCs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood using Biocoll (Merck) and Leucosep Tubes (Greiner, Bio-One), following standard protocols. Immune cell composition was analysed using flow cytometry and the following antibodies: FITC anti-human CD3, Pacific Blue™ anti-human CD4, APC/Cy7 anti-human CD8, PE anti-human CD197/CCR7, APC anti-human CD45RA antibody, PerCP/Cy5.5 anti-human CD279/PD-1 (all BioLegend), FITC TCR alpha/beta (eBioscience).
Primary human PBMCs were kept in RPMI 1640 medium supplemented with 10% FBS and 2 mM L-glutamine. Cells were incubated in the absence or presence of plate-bound anti-CD3 (OKT3; BioLegend) and anti-CD28 (CD28.2; BioLegend) antibodies for 120 h. All cells where then stimulated with PMA and ionomycin for 4 h and CD4+ T cells were collected using flow cytometry for RNA (gene expression) and DNA (methylation) analyses.
Some experiments were performed in previously reported genetically modified CD4⁺ Jurkat T cells (22 (link)). Jurkat T cells were kept in RPMI 1640 medium supplemented with 10% FBS and 2 mM l-glutamine. As indicated below, cells were incubated in the absence or presence of plate-bound anti-CD3 (OKT3; BioLegend) and anti-CD28 (CD28.2; BioLegend) antibodies for 24 h. Where indicated, cells were re-stimulated with PMA and ionomycin for 4 h.

Hofmann S.R., Carlsson E., Kapplusch F., Carvalho A.L., Liloglou T., Schulze F., Abraham S., Northey S., Russ S., Surace A.E., Yoshida N., Tsokos G.C, & Hedrich C.M. (2021). cAMP response element modulator α suppresses PD-1 expression and promotes effector CD4+ T cells in psoriasis. Journal of immunology (Baltimore, Md. : 1950), 207(1), 55-64.

+ Open protocol

+ Expand

Retroviral TCR Gene Transfer in PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

TCR gene transfer was carried out as described (54 (link)) with minor modifications. In brief, HEK-293 cells stably expressing GALV-env and MLV-gag/pol were grown to ~80% confluence and transfected with 3 μg of pMP71-TCR vectors in the presence of 10 μg Lipofectamine2000 (Life Technologies). At 48 and 72 h after transfection, 3 ml of retrovirus containing supernatant were harvested. 1 × 10⁶ human PBMCs, that had been frozen after isolation from healthy donors by ficoll gradient centrifugation, were thawed and stimulated with 5 μg/ml anti-CD3 (OKT3) and 1 μg/ml anti-CD28 (CD28.2) (Biolegend) coated plates in the presence of 300 U/ml recombinant human interleukin 2 (hIL-2) (Peprotech). Transductions at 48 and 72 h after stimulation were performed by addition of retrovirus containing supernatant and 4 μg/ml protamine sulfate followed by spinoculation for 90 min at 800 g and 32°C (1st transduction). For second transduction, retrovirus was preloaded onto retronectin (Takara)-coated plates followed by spinoculation for 30 min at 800 g and 32°C. Transduced PBMCs were maintained in the presence of 300 U/ml hIL-2 for a total of 2 weeks. At least 2 days prior to use in experiments, transduced PBMCs were cultured in the presence of 30 U/ml hIL-2.

Abdelaziz M.O., Ossmann S., Kaufmann A.M., Leitner J., Steinberger P., Willimsky G., Raftery M.J, & Schönrich G. (2019). Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation. Frontiers in Immunology, 10, 1776.

+ Open protocol

+ Expand

T Cell Activation and Antigen Presentation

Check if the same lab product or an alternative is used in the 5 most similar protocols

T cells were isolated from a leukoreduction system chamber from an HLA-A^∗02 positive healthy donor from the Stanford institutional blood bank using the RosetteSep human T cell enrichment cocktail (Stem Cell Technologies) and viably stored in liquid nitrogen. For T cell activation, T cells were thawed and stimulated with anti-CD3/CD28 beads (Life Technologies) in the presence of IL-2 (100 IU/mL). On days 1 and 2, activated T cells were retrovirally transduced using Retronectin (Takara) coated plates in media containing 100 IU/mL IL-2. anti-CD3/CD28 beads were removed on day 3 and media containing IL-2 were replenished once every 2 days. Following 8 days of in vitro expansion, T cells were co-cultured with 293A2 cells expressing full-length TMEM161A, EntS, LMP2, FluM1, or GFP alone at a 1:1 ratio. Following 18 h incubation, cells were stained with anti-CD3 (OKT3, Biolegend), anti-CD69 (FN50, Biolegend), anti-TCR⍺/β (IP26, Biolegend), anti-CD137 (4B4-1, BD Biosciences), and live/dead near-IR dye (Invitrogen). Data were acquired using FACS Fortessa (BD Biosciences) automated high throughput sampler and analyzed using FlowJo software (Treestar).

Chiou S.H., Tseng D., Reuben A., Mallajosyula V., Molina I.S., Conley S., Wilhelmy J., McSween A.M., Yang X., Nishimiya D., Sinha R., Nabet B.Y., Wang C., Shrager J.B., Berry M.F., Backhus L., Lui N.S., Wakelee H.A., Neal J.W., Padda S.K., Berry G.J., Delaidelli A., Sorensen P.H., Sotillo E., Tran P., Benson J.A., Richards R., Labanieh L., Klysz D.D., Louis D.M., Feldman S.A., Diehn M., Weissman I.L., Zhang J., Wistuba I.I., Futreal P.A., Heymach J.V., Garcia K.C., Mackall C.L, & Davis M.M. (2021). Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity, 54(3), 586-602.e8.

+ Open protocol

+ Expand

Single-Cell Sorting of Tumor-Infiltrating T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a 5-laser FACSAria Fusion sorter (Stanford FACS Facility) purchased using funds from the Parker Institute for Cancer Immunotherapy. Tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend) for viability assessment. This was followed by staining in PBS with 2% FBS in Fc Blocking solution (Biolegend) plus the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1(EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), anti-HLA-DR (L243, Biolegend). CD3⁺CD45⁺AquaZombie^- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at −80°C. Ectopic HLA-B^∗35 was detected with anti-HLA-BC monoclonal antibody (clone B1.23.2, Thermo Fisher Scientific). Transduced Jurkat 76 cells expressing exogenous TCRα/β chains were sorted on a FACSAria Fusion sorter at Stanford or a BD Biosciences Influx High Speed Cell Sorter at the Flow Cytometry Core Facility of the Cancer Institute of New Jersey.

+ Open protocol

+ Expand

Antibody Panel for Signal Transduction

Check if the same lab product or an alternative is used in the 5 most similar protocols

Anti-LAT, anti-PLC-γ1, and anti-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-NTAL NAP-07 monoclonal antibody was from EXBIO (Prague, Czech Republic). Antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, ZAP70, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, and anti-MEK were from Cell Signaling Technology. Anti-β-actin, anti-CD3 (OKT3), and anti-CD4 monoclonal antibodies were provided by Biolegend (San Diego, CA, USA).

Narbona-Sánchez I., Pérez-Linaza A., Serrano-García I., Vico-Barranco I., Fernández-Aguilar L.M., Poveda-Díaz J.L., Sánchez del Pino M.J., Medina-Varo F., Arbulo-Echevarria M.M, & Aguado E. (2023). Expression of Non-T Cell Activation Linker (NTAL) in Jurkat Cells Negatively Regulates TCR Signaling: Potential Role in Rheumatoid Arthritis. International Journal of Molecular Sciences, 24(5), 4574.

+ Open protocol

+ Expand

Generating CMV-Specific T Cell Lines and Clones

Check if the same lab product or an alternative is used in the 5 most similar protocols

CMV-specific T cell line (HD-2) and T cell clones (A1-8 and A1-9) specific for CMV pp65 protein were generated from a single HLA-A*0201+ healthy control. CMV-specific T cells were stained with HLA-PE-conjugated A*0201 CMV_NLVPMVATV Dextramer (Immunex, Copenhagen, Denmark) for 30 min at room temperature. HLA-A*0201-CMV-CTL were then sorted using a FACS Aria flow cytometer.
Sorted CMV-CTL from HD-2 cell line were then clonally selected using limiting dilution in T cell medium containing Serum-free medium (Cell-Genix, Freiburg, Germany), with 10% human AB serum (Innovative Research, MI) supplemented with IL-2 (1000 IU/ml), IL-15 (10ng/ml), IL-21 (10ng/ml), (Prospec, Ness-Ziona, Israel), penicillin (100IU/ml), streptomycin (100μg/ml) (Life Technologies, Carlsbad, CA), and amphotericin B (2.5mg/L) (Sigma-Aldrich, St Louis, MO) and seeded with anti-CD3 (OKT3) (Biolegend, CA, USA) and addition of γ-irradiated allogeneic feeder cells at a 5:1 E:T ratio. After 14 days, the clonal selection was repeated. Expanded T cell clones were transferred into larger vessels when required where they were subsequently expanded in T cell culture medium and maintained by biweekly stimulation with OKT3 and irradiated allogenic feeder cells.

Luo X.H., Poiret T., Liu Z., Meng Q., Nagchowdhury A, & Ljungman P. (2023). Different recovery patterns of CMV-specific and WT1-specific T cells in patients with acute myeloid leukemia undergoing allogeneic hematopoietic cell transplantation: Impact of CMV infection and leukemia relapse. Frontiers in Immunology, 13, 1027593.

+ Open protocol

+ Expand

ICAM-1-Fc Stimulation of PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs (1 ×10⁵) were incubated for 18–24 hrs with plate-bound ICAM-1-Fc (coated at 5 μg/ml) or with an isotype-matched negative control mAb (5 μg/ml MOPC-21, Sigma Aldrich). PBMCs were resuspended and Fc receptors were blocked by incubation in a solution of PBS containing 25% human AB serum (Atlanta Biologicals) for 20 minutes at 4°C, then cell-surface staining was performed for 30 minutes at 4 °C using the following human-specific antibodies or tetramers: anti-CD3 (OKT3, Biolegend), anti-CD4 (OKT4, Biolegend), anti-CD8α (HIT8a, Biolegend), anti-CD8β (SIDI8BEE, eBioscience), anti-CD11a (HI111, Biolegend), anti-CD56 (5.1H11, Biolegend), CD1d tetramer (PBS-57 loaded, NIH Tetramer Core Facility), MR1 tetramer (5-OP-RU loaded, NIH Tetramer Core Facility), anti-Vδ2 TCR (123R3, Miltenyi Biotec). Cells were fixed and permeabilized according to the manufacturer’s instructions using the BD Cytofix/Cytoperm kit (BD Biosciences), then stained with anti-IFN-γ (4S.B3, Biolegend), anti-PLZF (Mags.21F7, eBioscience), or the respective negative control mAbs suggested by the vendor. Cells were then washed, resuspended in PBS and analysed on an LSR II flow cytometer (BD Biosciences). Staining data were analyzed using FlowJo analysis software (Tree Star Inc).

Sharma A., Lawry S.M., Klein B.S., Wang X., Sherer N.M., Zumwalde N.A, & Gumperz J.E. (2018). LFA-1 Ligation by High Density ICAM-1 is Sufficient to Activate IFN-γ Release by Innate T lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 201(8), 2452-2461.

+ Open protocol

+ Expand

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells for flow cytometry analysis were harvested and stained with fluorescently labeled antibodies for 30 min at 4℃ in dark. Following antibodies were used: anti-CD80 (L307.4; BD Biosciences, San Diego, CA), anti-CD32 (FLI8.26; BD Biosciences), anti CD83 (HB15e; Biolegend, San Diego, CA), anti-CD137L (5F4; Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD8 (RPA-T8, Biolegend), anti-CD45RO (UCHL1, Biolegend), anti-CCR7 (G043H7, Biolegend), and anti-CD62L (DREG-26, BD Bioscience). pp65 Tetramer (Proimmune, Oxford, MC, UK) was used for tetramer staining and cells were analyzed with Canto (BD Biosciences). For analysis, lymphocyte population was gated based on FSC and SSC and further gating of CD8⁺ T cells were done by gating CD3 and CD8 double positive cells. Gating of memory differentiation was done either with CD45RO and CD62L or with CD45RO and CCR7.

Choi H., Lee H.J., Sohn H.J, & Kim T.G. (2023). CD40 ligand stimulation affects the number and memory phenotypes of human peripheral CD8+ T cells. BMC Immunology, 24, 15.

+ Open protocol

+ Expand

Activation and Expansion of CD8 T Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Peripheral blood mononuclear cells (PMBCs) were obtained from anonymous donors as previously described [23 (link)]. The CD8 T cells were isolated by negative selection using an enrichment kit for CD8 T cells (Stem Cell Technologies). These isolations yielded cells that were consistently >98% positive for CD8 T cells (data not shown). CD8 T cells were activated for 5 days with magnetic Dynabeads (Invitrogen) bound with anti-CD3 (OKT3, BioLegend) and anti-CD28 (CD28.2, BioLegend) antibodies in the presence of 100 U/mL IL-2. Following activation and expansion, the stimulatory anti-CD3/anti-CD28 beads and IL-2 were removed, and the cells were rested for 24 h in complete RPMI (RPMI 1640 supplemented with 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mM l-glutamine (Gibco)) before further experimentation. Activated CD8 T cells were split into different flasks and exposed to different doses of recombinant cytokines (R&D Systems) or exposed to IL-12 for different times. Media was not changed in between the times. Cells were then washed three times in complete RPMI to remove the recombinant cytokines.

Vacaflores A., Freedman S.N., Chapman N.M, & Houtman J.C. (2016). Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production. Molecular immunology, 81, 1-15.

+ Open protocol

+ Expand

Evaluating CD8+ T-cell Proliferation in HNSCC

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD3⁺ T cells were purified from healthy donor human PBMCs using the EasySep Human CD3 Positive Selection Kit II (StemCell Technologies, catalog no. 17851) according to the manufacturer's protocol. Purified CD3⁺ T cells were stained with 5 µmol/L CFSE (Invitrogen, catalog no. C34554) and resuspended in complete RPMI media. CD3⁺ T cells (1 × 10⁵) were cocultured with varying ratios of HNSCC cells and 5 µg/mL anti-CD3 (OKT3, BioLegend) and 10 µg/mL anti-CD28 (CD28.2, BioLegend) stimulation with/without 10 µg/mL CDA in 100 µL in round-bottom 96-well plates. Following a 4-day incubation at 37°C and 5% CO₂, T cells were stained with APC-conjugated CD8 (SK1, BioLegend) and CD8⁺ T-cell proliferation was assessed by flow cytometry using the Attune NxT Focus cytometer (Thermo Fisher Scientific) and data were analyzed with FlowJo software.

Saulters E.L., Kennedy P.T., Carter R.J., Alsufyani A., Jones T.M., Woolley J.F, & Dahal L.N. (2024). Differential Regulation of the STING Pathway in Human Papillomavirus–Positive and -Negative Head and Neck Cancers. Cancer Research Communications, 4(1), 118-133.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!