Plate bound anti cd3

Manufactured by BioLegend

Sourced in United States

Plate-bound anti-CD3 is a laboratory product that binds to the CD3 receptor on T cells. It is used to activate and stimulate T cell proliferation and function in experimental settings.

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Close spelling variants of this product

Plate-bound anti-CD3 (OKT3 - (2 citations) Plate-bound anti-CD3 antibody (clone OKT3 – (2 citations) Plate bound anti-CD3 antibody (3 citations) Anti-CD3 (468 citations)

8 protocols using plate bound anti cd3

CFSE-Based Proliferation Assay for CD4+ T Cells

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For the proliferation assay, 1 × 10⁶ purified CD4+CD25– T cells were labeled with the carboxyfluorescein succinimidyl ester (CFSE) (1 μM; Invitrogen, Carlsbad, CA, USA) in 1 mL pre-warmed phosphate-buffered saline, followed by incubation for 10 min at room temperature and neutralization with pre-chilled complete medium containing 10% fetal bovine serum. CFSE-labeled cells were re-suspended and cultured in complete medium in the presence of plate-bound anti-CD3 (coating concentration was 2.5 μg/mL, Biolegend, San Diego, CA, USA) and soluble anti-CD28 (2.5 μg/mL, Biolegend, San Diego, CA, USA) in 24-well flat-bottom plates for 72 h. After culture, the cells were collected for anti-TIGIT APC and anti-CD226 PerCP-CY5.5 staining and then analyzed to determine the CFSE intensities. Each experiment was performed and analyzed using a FACS JAZZ instrument.

Li W., Deng C., Yang H., Lu X., Li S., Liu X., Chen F., Chen L., Shu X., Zhang L., Liu Q., Wang G, & Peng Q. (2021). Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis. Arthritis Research & Therapy, 23, 15.

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MAIT Cell Activation by E. coli-loaded Monocytes

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Healthy adult blood was obtained from the blood bank of the University Leipzig, approved by the local ethics committee (Ref. #079-15-09032015). Peripheral blood mononuclear cells were isolated from male human donors at age 20-50 by Ficoll-Paque™ density-gradient (GE Healthcare, UK) centrifugation. CD161+ TCR Vα7.2+ MAIT cells and CD14+ monocytes were obtained by positive magnetic separation using respective microbeads (Miltenyi Biotec, Germany). For RNA-Seq experiments, monocytes were loaded with fixed E. coli at a MOI of 10 for 3h, followed by intense washing in PBS to remove E. coli. MAIT cells and E. coli-loaded monocytes were incubated at a ratio of 1:1 for 16h. In some experiments, MAIT cells were stimulated with 50ng/ml IL-12 and 50ng/ml IL-18 (both from MBL International, MA, USA), 10µg/ml plate-bound anti-CD3 and 1µg/ml soluble anti-CD28 (both from Biolegend, CA, USA) or a combination of both for 16h. For TWIST1 inhibition experiments, the small molecule inhibitor harmine (Sigma-Aldrich, Germany) was used at a concentration of 20 or 40µM, DMSO served as solvent control.

Schubert K., Karkossa I., Schor J., Engelmann B., Steinheuer L.M., Bruns T., Rolle-Kampczyk U., Hackermüller J, & von Bergen M. (2021). A Multi-Omics Analysis of Mucosal-Associated-Invariant T Cells Reveals Key Drivers of Distinct Modes of Activation. Frontiers in Immunology, 12, 616967.

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Naive CD4+ T Cell Differentiation

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Naïve CD4+ T cells were isolated from the spleen of 6–8 w/o female mice by magnetic cell sorting (Biolegend, CA, USA). Magnetically sorted naïve CD4+ T cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution at 37°C in an incubator with 5% CO₂. Plate-bound anti-CD3 (2 μg/mL, Biolegend) and soluble anti-CD28 (1 μg/mL, Biolegend) were used to activate naïve CD4+ T cells, followed by differentiation into Th1 (IL-12 [5 ng/mL; Peprotech, NJ, USA], IL-2 [(50 U/mL); Biolegend] and anti-IL-4 [10 μg/mL; Peprotech]); Th2 (IL-4 [20 ng/mL; Biolegend] and anti-IFN-γ [10 μg/mL; Biolegend]), Th9 (IL-4 [20 ng/mL], TGF-β [2 ng/mL; Biolegend] and anti-IFN-γ [10 μg/mL]), Th17 (TGF-β [2 ng/mL], IL-6 [100 ng/mL; Peprotech], IL-1β [10 ng/mL; Peprotech], IL-23 [10 ng/mL; Peprotech], anti-IFN-γ [10 μg/mL] and anti-IL-4 [10 μg/mL]) and Treg cell differentiating conditions (TGF-β [2 ng/mL], anti-IL-4 [10 μg/mL; Peprotech] for 3 days. The cultures were expanded for further 2 days by adding three times of fresh media for all the culture conditions with IL-4 and TGF-β for Th9, half the concentration of IL-6, IL-1β, IL-23 for Th17 and IL-2 for Treg conditions. Calcitriol used in the experiments was procured from Sigma Aldrich (MO, USA).

Vyas S.P., Hansda A.K., Kaplan M.H, & Goswami R. (2020). Calcitriol regulates the differentiation of IL-9-secreting Th9 cells by modulating the transcription factor PU.1. Journal of immunology (Baltimore, Md. : 1950), 204(5), 1201-1213.

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Modulation of T Cell Differentiation by DC-Derived Factors

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BM-DCs were stimulated with 1 μg ml⁻¹ LPS, and CM was collected after 18 h. CD4⁺ T cells were purified from naive W/T mice and activated with plate-bound anti-CD3 (2 μg ml⁻¹; Biolegend) and soluble anti-CD28 (5 μg ml⁻¹; eBioscience) in the presence of recombinant TGF-β1 (5 ng ml⁻¹, PeproTech). CM from W/T or Itgam^−/− DCs was added to the T-cell culture at a ratio of 1:1. On day 3, the cells were restimulated with 10 ng ml⁻¹ of PMA (Sigma) and 250 ng ml⁻¹ of Ionomycin (Calbiochem) in the presence of GolgiStop (BD Pharmingen) for 5 h. Cells were then intracellularly stained with PE-conjugated anti-IL-17 (TC11-18H10; dilution 1:100) and FITC-labelled anti-IFN-γ (XMG1.2; dilution 1:100) (BD Pharmingen). For the detection of T regulatory cells, cells were intracellularly stained with antigen-presenting cell-conjugated Foxp3 (FJK-16s; dilution 1:100) (eBioscience) before the PMA/Ionomycin re-stimulation.

Ling G.S., Bennett J., Woollard K.J., Szajna M., Fossati-Jimack L., Taylor P.R., Scott D., Franzoso G., Cook H.T, & Botto M. (2014). Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages. Nature Communications, 5, 3039.

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Induction of Regulatory T Cells

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Magnetically isolated naïve CD4⁺ CD25^Neg T cells isolated from the spleen were incubated for 72 hours in complete media (RPMI-1640 + L-glutamine, Corning Inc. Corning NY, USA; supplemented with 10 % FBS, 100 IU penicillin and 100 μg/ml streptomycin; Invitrogen, Carlsbad, CA, USA) in the presence of IL-2 (5 IU/ml;), plate-bound anti-CD3 (1 μg/ml; BioLegend, San Diego, CA, USA), soluble anti-CD28 (37.15; 1 μg/ml; BioLegend, San Diego, CA, USA) and TGFβ (5ng/ml; R&D Systems, Minneapolis, MN, USA). The expression of FoxP3 was measured by flow cytometry.

Collins C.B., Nguyen T.T., Leddy R.S., Alula K.M., Yeckes A.R., Strassheim D., Aherne C.M., Luck M.E., Karoor V., Jedlicka P., Pierce A, & de Zoeten E.F. (2023). Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation. Mucosal immunology, 17(1), 94-110.

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Transcriptional Profiling of Activated CD4 T Cells

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In all, 2000 naive CD4 T cells were isolated by FACS, sorting directly into lysis buffer (RLT buffer (Qiagen)) for timepoint 0 h. At the same time, FACS-sorted naive CD4 T cells (10⁶ cells/mL) were activated in vitro using a combination of plate-bound anti-CD3 (2 μg/mL) (BioLegend) and soluble anti-CD28 (2 μg/mL) (BioLegend) antibodies for 24 h before 2000 live cells were FACS sorted directly into lysis buffer (RLT buffer (Qiagen)). RNA was extracted using the RNeasy Plus Micro kit (Qiagen). RNA was processed using the RNA-Seq Poly A method and 100 bp paired-end RNA-Seq was performed on the Illumina HiSeq4000 platform (Wellcome Trust Centre for Human Genetics, University of Oxford). Sequencing data were trimmed using Trimmomatic and aligned to the mm10 genome using STAR (version 2.5.3a)^{70 (link)}. Reads were assigned to genes using HTSeq (intersection non-empty)^{71 (link)}. Differential analysis was performed using edgeR^{72 (link)}. Significant genes were defined as those with Benjamini-Hochberg adjusted p-values less than 0.05. ClusterProfiler was used for gene ontology (GO) analysis^{73 (link)}.

Oftedal B.E., Maio S., Handel A.E., White M.P., Howie D., Davis S., Prevot N., Rota I.A., Deadman M.E., Kessler B.M., Fischer R., Trede N.S., Sezgin E., Maizels R.M, & Holländer G.A. (2021). The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function. Communications Biology, 4, 681.

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T Cell Activation and IL-2 Stimulation

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The isolated primary CD4⁺ T cells were cultured in RPMI1640 medium supplemented with 10% FBS/penicillin-streptomycin in a 37 °C incubator and activated by 2 μg/ml of plate-bound anti-CD3 (Biolegend; 317353) and 1 ug/ml of soluble anti-CD28 (Biolegend; 302901) for 72 h.
IL-2-stimulation: The above TCR-activated T cells were rested overnight. Two hundred International Units of IL-2 were added to the cells for 4 h.

Chai H., Tjong H., Li P., Liao W., Wang P., Wong C.H., Ngan C.Y., Leonard W.J., Wei C.L, & Ruan Y. (2023). ChIATAC is an efficient strategy for multi-omics mapping of 3D epigenomes from low-cell inputs. Nature Communications, 14, 213.

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CFSE-based T Cell Activation Assay

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Thawed PBMCs were rested overnight in media supplemented with 10 % fetal bovine serum. Cells were washed and 1 × 10⁶ cells were labeled with 1 mL of 5 μM CFSE (BioLegend) following an established protocol reported elsewhere [41 (link)]. CFSE-labeled PBMCs were incubated in a 96-well culture plate and stimulated with media, uRBCs, iRBCs, and plate-bound anti-CD3 (BioLegend) at a density of 2.5 × 10⁵ cells per condition. As before, an effector-to-target ratio of 1:3 was used with uRBCs and iRBCs. Anti-CD28 and anti-CD49d were added for costimulation (3 μg/mL BioLegend). At day 7, supernatants were collected and frozen for downstream cytokine analysis and the cells washed and stained with surface antibodies (Brilliant Violet 421-conjugated anti-CD4, APC-conjugated anti-CCR7, APC-Cy7-conjugated anti-CD3 (BioLegend), PE-Cy7-conjugated anti-CD27, and PerCP-Cy5.5-conjugated anti-CD8 (BD Pharmingen)) before acquisition. Once again, LIVE/DEAD aqua amine was included to exclude dead cells from downstream analysis.

Bediako Y., Ngoi J.M., Nyangweso G., Wambua J., Opiyo M., Nduati E.W., Bejon P., Marsh K, & Ndungu F.M. (2016). The effect of declining exposure on T cell-mediated immunity to Plasmodium falciparum – an epidemiological “natural experiment”. BMC Medicine, 14, 143.

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