Fitc conjugated anti cd3

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

FITC-conjugated anti-CD3 is a fluorescent-labeled antibody that binds to the CD3 protein on the surface of T cells. It can be used to identify and isolate T cells in flow cytometry and cell sorting applications.

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Close spelling variants of this product

Anti-CD3 (512 citations) CD3-FITC (66 citations) Anti-CD3-FITC (50 citations) FITC conjugated anti-mouse CD3 (3 citations) Anti-CD3 fluorescein isothiocyanate (FITC)-conjugated antibody (3 citations) FITC‐conjugated CD3 (2 citations)

15 protocols using fitc conjugated anti cd3

Immunophenotyping of Lymphocyte Subsets

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Cells were doubly incubated with FITC-conjugated anti-CD45R (1 mg/10 6 cells; eBioscience, Thermo Fisher Scientific, Waltham, MA) and PE-conjugated anti-IgM (1 mg/10 6 cells; BD Biosciences, Heidelberg, Germany), FITC-conjugated anti-CD45R (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD138 (1 mg/10 6 cells; Abcam, Cambridge, UK), FITC-conjugated anti-CD3 (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD4 (0.5 mg/10 6 cells; eBioscience) as well as FITC-conjugated anti-CD3 (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD8 (0.125 mg/10 6 cells; eBioscience), respectively. The percentages of
T cells were determined through a lymphocyte gate by flow cytometry (BD Accuri C6, BD Biosciences).

Li J., Basler M., Alvarez G., Brunner T., Kirk C.J, & Groettrup M. (2018). Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation. Kidney international, 93(3).

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Synthesis and Characterization of PLGA-b-PEG-b-PLGA Copolymer

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PLGA-b-PEG-b-PLGA triblock copolymer (number-average molecular weight (M_{n, NMR}) = 4200 g/mol, LA/GA = 75:25, mol/mol) was synthesized as our previous works [30 (link)]. Percoll density gradient, 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and anti-collagen type II antibody (anti-COL-II Ab) enzyme-linked immunosorbent assays (ELISAs) kit were purchased from Sigma-Aldrich, USA. Chick type II collagen (COL-II) and complete Freund's adjuvant (CFA) were acquired from Chondrex, USA. FITC-conjugated anti-CD3, PE-conjugated anti-CD4, and APC-conjugated anti-CD8 mAbs were obtained from eBioscience, San Diego, CA. Dimethyl sulfoxide (DMSO) was stored over calcium hydride (CaH₂) and purified by vacuum distillation with CaH₂.

Liu H., Ding J., Wang J., Wang Y., Yang M., Zhang Y., Chang F, & Chen X. (2015). Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells. PLoS ONE, 10(3), e0120596.

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Immunophenotyping of T-cell subsets

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For surface staining, single cell suspensions obtained as above were stained for 30 min on ice with the following fluorescence conjugated antibodies including FITC-conjugated anti-CD3, APC-conjugated anti-CD4 and PE-conjugated anti-CD25 from eBioscience. For intracellular staining, cell suspensions after surface staining were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (eBioscience) as per the manufacturer’s instructions. Then the fixed/permeabilized cells were stained with Percp-Cy5.5-conjugated anti-Foxp3 (eBioscience) for 45 min on ice. The stained cells were analysed by flow cytometry on a FACS calibur (Becton Dickinson) using CellQuest analysis software (BD Biosciences).

Gao S.X., Sun J., Li H.H., Chen J., Kashif M.R., Zhou P., Wei L., Zheng Q.W., Wu L.G, & Guan J.C. (2020). Prenatal exposure of staphylococcal enterotoxin B attenuates the development and function of blood regulatory T cells to repeated staphylococcal enterotoxin B exposure in adult offspring rats. Journal of Medical Microbiology, 69(4), 591-599.

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Immune Cell Characterization Protocols

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RPMI 1640 medium, α-MEM, trichloroacetic acid (TCA), Ehrlich’s reagent and 1-methyl-DL-tryptophan (1-MT) were from Sigma-Aldrich (St. Louis, MO). Anti-IDO monoclonal antibody (clone 10.1) was from Upstate Cell Signaling Solutions (Lake Placid, NY). FBS was from Invitrogen (Carlsbad, CA). Recombinant mouse and human TNFα, IL-1β, IFNγ, and IL-2 were from R&D Systems (Minneapolis, MN). Anti-CD3 and anti-CD28 monoclonal antibodies were from R&D Systems. G418 was from Invitrogen. FITC-conjugated anti-CD3, PE-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-Cy5-conjugated anti-CD8, FITC-conjugated anti- F4/80, PE-conjugated anti-NKG2D, and their respective isotypes were all from eBioscience (La Jolla, CA).

Ling W., Zhang J., Yuan Z., Ren G., Zhang L., Chen X., Rabson A.B., Roberts A.I., Wang Y, & Shi Y. (2014). Mesenchymal Stem Cells Employ IDO to Regulate Immunity in Tumor Microenvironment. Cancer research, 74(5), 1576-1587.

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Quantification of T Cell Subsets

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After treatment for 24 h at 37°C, FITC-conjugated anti-CD3 (eBioscience, Inc, San Diego, CA, USA), APC-conjugated anti-CD4 (eBioscience), and PE-conjugated anti-CD8 (eBioscience) were incubated with the cells of each group for 1 h at 4°C. Afterwards, the cells were fixed with 1% paraformaldehyde and washed. The percentages of CD3⁺CD4⁺ and CD3⁺CD8⁺ were calculated using a flow cytometer (BD Biosciences, Franklin lakes, NJ, USA).

Jiang Y., Li T., Qian Y., Zuo X, & Liu J. (2022). Morphine in Combination with Ketamine Improves Cervical Cancer Pain and Suppresses Immune Function via the JAK3/STAT5 Pathway. Pain Research & Management, 2022, 9364365.

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Multiparametric Flow Cytometry Analysis

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FITC-conjugated anti-CD3, APC-conjugated anti-CD4, APC-conjugated anti-CD8, and their corresponding isotype controls were purchased from eBioscience (San Diego, CA). PE-Cy5-conjugated anti-CD4, PE-Cy5-conjugated anti-CD8, FITC-conjugated anti-CD44, and their corresponding isotype controls, were obtained from BioLegend (San Diego, CA). PE-conjugated anti-CD45RB, PE-conjugated anti-Fas, and their corresponding isotype controls were purchased from BD Bioscience (San Jose, CA). FITC-conjugated anti-CD28 and its corresponding isotype control were purchased from Serotec (Oxfordshire, UK). Rabbit polyclonal primary anti-Ki-67 and its isotype control were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. PerCP-conjugated donkey anti-rabbit IgG antibody was obtained from Santa Cruz Biotechnology. T cell mitogen concanavalin A (Con A) was obtained from Sigma (St. Louis, MO).

Sheu T.T., Chiang B.L., Yen J.H, & Lin W.C. (2014). Premature CD4+ T Cell Aging and Its Contribution to Lymphopenia-Induced Proliferation of Memory Cells in Autoimmune-Prone Non-Obese Diabetic Mice. PLoS ONE, 9(2), e89379.

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Flow Cytometry Analysis of Immune Cells

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For flow cytometry, the following antibodies for the analysis of human samples were used: FITC-conjugated anti-CD16, PE-conjugated anti-PD-L1, PerCP-conjugated anti- CD45, APC-conjugated anti-CD11b, APC-conjugated anti-CD14, FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PerCP-conjugated anti-CD4 and APC-conjugated anti-PD-1. All human antibodies were purchased from BD Bioscience (San Jose, USA). Antibodies used in the mouse experiments were purchased from eBioscience (San Diego, CA) as follows: FITC-conjugated anti-Gr-1, PE-conjugated anti-PD-L1, PerCP-conjugated anti-CD11b, APC-conjugated anti-CD48, APC-conjugated anti-CD8, FITC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PerCP-conjugated anti-CD4. Data were analysed with FlowJo5.6.7 (Tree star, Inc., Ashland, OR, USA).

He G., Zhang H., Zhou J., Wang B., Chen Y., Kong Y., Xie X., Wang X., Fei R., Wei L., Chen H, & Zeng H. (2015). Peritumoural neutrophils negatively regulate adaptive immunity via the PD-L1/PD-1 signalling pathway in hepatocellular carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 34, 141.

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Multiparametric Flow Cytometry Analysis

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The following mAbs were used in this study: anti-CCR1 (R&D System, MAB 145-100, Minneapolis U.S.A.), anti-CCR5 (R&D System, MAB 182-100 Minneapolis U.S.A.), anti-CCR7 (R&D System, MAB 197-100 Minneapolis U.S.A.), anti-CXCR1/IL-8 RA (R&D System, MAB 173-100 Minneapolis U.S.A.), anti-CXCR3 (R&D System, MAB 160-100), anti-CXCR4 (R&D System, MAB 173-100), PE-conjugated anti-CX₃CR1 (Medical & Biological Laboratories Co., LTD, D070-5), FITC-conjugated anti-CD3 (eBioscience, 11-0038-42 Thermofisher scientific, Waltham, Massachusetts, Stati Uniti), PE-cyanine 7-conjugated anti-CD56 (Beckman Coulter, A21692, Brea, California U.S.A.), PE-conjugated anti-CD16 (130-106-704, Miltenyi Biotec Bergisch Gladbach, Germany). The staining with the appropriate unlabeled mAbs are followed by PE-conjugated isotype-specific goat anti-mouse second reagent (Southern Biotechnology Associated, Birmingham, AL, U.S.A.), and fluorescence was quantified on a Gallios™ Flow Cytometer (Beckman Coulter, Brea, California U.S.A.).

Parodi M., Raggi F., Cangelosi D., Manzini C., Balsamo M., Blengio F., Eva A., Varesio L., Pietra G., Moretta L., Mingari M.C., Vitale M, & Bosco M.C. (2018). Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration. Frontiers in Immunology, 9, 2358.

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Multiparametric Immune Profiling Protocol

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Fluorescein isothiocyanate (FITC)-conjugated anti-CD38, Alexa 488-anti-CXCR5, Alexa F-647-anti-CXCR5, phycoerythrin (PE)-conjugated anti-IL-21, PE-Cy7-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-IL-6 and FITC-conjugated anti-IFN-gamma were purchased from BD Bioscience and BD Pharmingen™ (San Diego, CA, USA). FITC-conjugated anti-CD3, PE-conjugated anti-ICOS, PE-conjugated anti-IL-17a, peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 and anti-CD8 and APC-conjugated anti-CD279 (PD-1) were purchased from eBioscience (San Diego, CA, USA); and APC-Cy7-conjugated anti-CD3 and anti-CD27, PE-conjugated anti-CD24, PE-Cy7-conjugated anti-CD19 and PerCP-Cy5.5-conjugated anti-IgD were purchased from BioLegend (San Diego, CA, USA). Peripheral blood mononuclear cells (PBMCs) were isolated and phenotypic analysis performed using optimal concentrations of mAbs according to standard protocols (25 (link), 26 (link)). Aliquots of cells were also utilized for flow cytometry and FlowJo software analysis (Tristar Inc, San Carlos, CA, USA). At least 50,000 events per sample were analyzed.

Wang L., Sun Y., Zhang Z., Jia Y., Zou Z., Ding J., Li Y., Xu X., Jin L., Yang T., Li Z., Sun Y., Zhang J.Y., Lv S., Chen L., Li B., Gershwin M.E, & Wang F.S. (2015). CXCR5+ CD4+ T Follicular Helper Cells Participate in the Pathogenesis of Primary Biliary Cirrhosis. Hepatology (Baltimore, Md.), 61(2), 627-638.

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Antibody Reagents for T-Cell Activation

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Mouse anti-CD3 (OKT-3) antibody was purchased from e-Bioscience (eBioscience, San Diego, CA, USA). Anti-Nck1, anti-phospho-MEK1/2 and anti-phospho-p38, antibodies were from Cell Signaling (Cell Signaling Technology, Danver, MA, USA). Anti-Nck2 antibody was purchased from Abnova (Abnova Corp., Taipei, Taiwan). Anti-phospho-Erk1/2 antibody was from Upstate (Upstate Biotechnology, Lake Placid, NY, USA). Anti-phospho IκBα antibody was supplied from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). FITC-conjugated anti-CD3 and PE-conjugated anti-CD69 antibodies were purchased from eBioscience (eBioscience, San Diego, CA, USA).

Ngoenkam J., Paensuwan P., Preechanukul K., Khamsri B., Yiemwattana I., Beck-García E., Minguet S., Schamel W.W, & Pongcharoen S. (2014). Non-overlapping functions of Nck1 and Nck2 adaptor proteins in T cell activation. Cell Communication and Signaling : CCS, 12, 21.

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