Cd3 apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD3-APC is a fluorochrome-conjugated antibody that binds to the CD3 receptor, a component of the T-cell receptor complex. It is used for the identification and enumeration of T cells in flow cytometry analysis.

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Close spelling variants of this product

Anti-human CD3/APC-eFluor780 (3 citations) Anti-human CD3-APC-Cy7 (2 citations) Anti-human CD3-APC (3 citations) Allophycocyanin (APC)-Cy7-conjugated anti-human CD3 (2 citations) Anti-CD3-APC (clone 17A2, (2 citations) CD3-APC efluor780 (SK7, (3 citations) APC-Cy7-conjugated anti-human CD3 (2 citations) CD3-APC (clone SK7, (2 citations) CD3-APC (145-2C11), (4 citations) CD3-APC-Cy7 (10 citations)

57 protocols using cd3 apc

Comprehensive Immune Cell Profiling Protocol

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The cells were stained with monoclonal antibodies to T-cell phenotype CD3 APC, CD4 PerCP Cy 5.5, CD8 APC Cy7, CD27 FITC, CD45RA PE (eBioscience, CA, USA). The cells were also stained with monoclonal antibodies to MDSC phenotype CD3 APC, CD19 APC, CD56 APC, HLA-DR APC e-fluor 780, CD33 PerCP Cy5.5, CD11b PE, CD14 PE Cy7, CD15 FITC (eBioscience, CA, USA). After 30 min of incubation with monoclonal antibodies, in the dark and at 4°C, the cells were washed with PBS and centrifuged. Living cells (based on forward and side scatter) were acquired in the FACS Canto II using the DIVA software (Becton Dickinson, USA). Further analyses of FACS data were performed using the 9.3 FLOWJO software (Tree Star, USA).
T lymphocytes were characterized as described previously (36 (link)).

Naïve: CD3⁺CD4⁺CD45RA⁺CD27⁺ or CD3⁺CD8⁺CD45RA⁺CD27⁺ (Naïve).

Central memory: CD3⁺CD4⁺CD45RA⁻CD27⁺ or CD3⁺CD8⁺CD45RA⁻CD27⁺ (CM).

Effector memory: CD3⁺CD4⁺CD45RA⁻CD27⁻ or CD3⁺CD8⁺CD45RA⁻CD27⁻ (EM).

Effector memory re-expressing CD45RA: CD3⁺CD4⁺CD45RA⁺CD27⁻ or CD3⁺CD8⁺CD45RA⁺CD27⁻ (EMRA).

Myeloid-derived suppressor cells were characterized as:

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD15⁺ granulocytic or

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD14⁺ monocytic.

Alves A.S., Ishimura M.E., Duarte Y.A, & Bueno V. (2018). Parameters of the Immune System and Vitamin D Levels in Old Individuals. Frontiers in Immunology, 9, 1122.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry analysis was performed on a CytoFLEX S flow cytometer (Beckman Coulter).
For blood and brain macrophage and eosinophil assessment, PBMCs and BMNCs were isolated as previously described [25 (link)] and then incubated with Siglec F PE (BD), CD11c PE-Cyanine5 (eBioscience), or CD11b APC-cy7 (eBioscience) at 4 °C for 30 min.
To determine the cell sources of Chi3l3, BMNCs were stained with CD11b PE (eBioscience), F4/80 PE-Cyanine5 (eBioscience), CD45 PE-eFluor 610 (eBioscience), Ly-6C PerCP/Cy5.5 (Biolegend), CX3CR1 Alexa Fluor 488 (eBioscience), or CCR2 Phycoerythrin (R&D) at 4 °C for 30 min.
For measurement of IL-5 and IL-13 levels, spleen cells [30 (link)] from normal and AC-infected mice were incubated with CD3 APC (eBioscience), CD4 FITC (eBioscience), and IL-13 PE cy7 (eBioscience) at 4 °C for 30 min and analyzed by flow cytometry.
To determine the possible effects of Chi3l3 and sAg on IL-13 production, spleen cells isolated from normal and AC-infected mice were cultured in 24-well cell culture plates for 72 h, followed by a 12-h incubation with Brefeldin A and 6-h stimulation with PMA and ionomycin. Then, the cells were incubated with CD3 APC (eBioscience), CD4 FITC (eBioscience), or IL-13 PE cy7 (eBioscience) at 4 °C for 30 min and analyzed by flow cytometry.

Wan S., Sun X., Wu F., Yu Z., Wang L., Lin D., Li Z., Wu Z, & Sun X. (2018). Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis. Journal of Neuroinflammation, 15, 31.

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CAR T Cell Immunophenotyping and Transduction

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Immunophenotyping of the apheresis was conducted with the following fluorophore-conjugated antibodies: CD3-phycoerythrin (PE) (Beckman Coulter), CD8-PE-Cy7 (Invitrogen), CD14-allophycocyanin (APC) (eBioscience), and CD45-fluorescein isothiocyanate (FITC) (Beckman Coulter). In-process transduction efficiency of the CD19-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and biotinylated goat-anti-mouse Fab (Jackson Immunoresearch Lab) followed by PE-conjugated streptavidin (MP Biomedicals). In-process transduction efficiency of the BCMA-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and BCMA-Fc-APC.¹² (link) The effector memory and central memory immunophenotyping was conducted using the following monoclonal antibodies: CD27-APC, CD28-FITC, CD62L-FITC, CCR7-FITC, CD45RA-APC (Invitrogen), and CD127-eFlour450 (eBioscience). Dead cells were excluded from analysis using either 7AAD or DAPI staining. Flow data acquisition was performed on an LSRII (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star).

Wang X., Borquez-Ojeda O., Stefanski J., Du F., Qu J., Chaudhari J., Thummar K., Zhu M., Shen L.B., Hall M., Gautam P., Wang Y., Sénéchal B., Sikder D., Adusumilli P.S., Brentjens R.J., Curran K., Geyer M.B., Mailankhody S., O’Cearbhaill R., Park J.H., Sauter C., Slovin S., Smith E.L, & Rivière I. (2021). Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing. Molecular Therapy. Methods & Clinical Development, 22, 377-387.

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Disulfiram and Copper Gluconate Protocol

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Disulfiram (Selleck, S1680) and copper gluconate (CuGlu, Sangon Biotech, A503014) with over 99% purity were used for the study. The antibody to NPL4 (sc-365796) was obtained from Santa Cruz. The antibodies to EIF2S1 (ab32157), XBP1 (ab220783), and EIF2S1 (phosphor S51) (ab32157) were obtained from Abcam. The antibodies to CHOP (A0221), Ubiquitin (A18185), β-tubulin (A7074), and Lamin-B (A19970) were obtained from Abclonal. The antibodies to Zombie NIR™ APC-CY7(423106), CD45 FITC (103108)/PE (103106), CD11c PE (117308)/APC (117310), I-Ab PE-CY7 (116420), CD80 PE-CY5.5 (104712), CD86 APC (105012), CD3 APC (100236), CD8 PE (100708)/APC-CY7 (100712), CD4 PE (130310), CD44 PE-CY7(103010), CD62L APC (104412), Granzyme B PE-CY7(372213), and IFN-γ PE-CY5.5(505821) were obtained from eBioscience (San Diego, CA, USA).

Gao X., Huang H., Pan C., Mei Z., Yin S., Zhou L, & Zheng S. (2022). Disulfiram/Copper Induces Immunogenic Cell Death and Enhances CD47 Blockade in Hepatocellular Carcinoma. Cancers, 14(19), 4715.

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Peripheral Immune Cell Profiling in TBI

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Peripheral immune cell infiltration was analyzed as described previously [19 (link)]. Briefly, blood and brain were collected from Sham or TBI mice at 3 days following TBI. Mice were euthanized and peripheral blood was obtained by cardiac puncture. And then mice were perfused with cold Hank’s balanced salt solution (HBSS) to collect the impacted hemisphere. The impacted hemispheres were digested with Neural Tissue Dissociation Kit (Miltenyi) using gentleMACS Octo Dissociator with heaters (Miltenyi). The resulting homogenate was passed through a 70 μm cell strainer, and Percoll was added to create a 30% and 70% percoll gradient with distinct separation lines. After centrifugation and removal of debris, the precipitate was resuspended. Then cells were stained at 4 ℃ in darkness for 30 min with the following fluorophore-conjugated antibodies: CD11b-APC-eFlour 780 (eBioscience, 47–0112-82, 1:200), CD45-eFlour 450 (eBioscience, 48–0451-82, 1:50), Ly6G(Gr1)-PE(eBioscience, 12–9669-82, 1:200), F4/80-PE (eBioscience, 12–4801-82, 1:200), CD11c-PerCP cy5.5(eBioscience, 45-0114-82, 1:100) Ly-6G-FITC (Thermo Fisher Scientific, 11-9668-82, 1:200), CD3-APC (eBioscience, 17–0032-82, 1:200), CD19-FITC (BD Bioscience, 561740, 1:200). Flow cytometry was performed using the Beckman CytoFlex, and the obtained data were analyzed using FlowJo software.

Meng S., Cao H., Huang Y., Shi Z., Li J., Wang Y., Zhang Y., Chen S., Shi H, & Gao Y. (2023). ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury. Journal of Neuroinflammation, 20, 244.

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Isolation and Expansion of Antigen-Specific CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll underlay, resuspended in T-cell media (RPMI, 10% pooled human serum, 1% penicillin-streptomycin, 1% l-glutamine) at 4 × 10⁶ cells/mL, and stimulated with peptides (20 µg/mL total) in 48-well plates for 14 days, with addition of medium and IL-2 starting on day 7. Cells were stained with individual tetramers for 75 min at 37°C, followed by CD4 PerCP (BD Biosciences), CD3 APC (eBioscience), and CD25 FITC (BioLegend) for 15 min at 4°C run on a FACSCalibur (BD Biosciences), and analyzed using FlowJo (Treestar Inc.). Clones were isolated by sorting single tetramer–positive CD4⁺ T cells using a FACSAria (BD Biosciences) and expanded in 96-well plates in the presence of 1 × 10⁵ irradiated PBMC and 2 µg/mL phytohemagglutinin (Remel), with addition of media and IL-2 starting on day 10.

Arribas-Layton D., Guyer P., Delong T., Dang M., Chow I.T., Speake C., Greenbaum C.J., Kwok W.W., Baker R.L., Haskins K, & James E.A. (2020). Hybrid Insulin Peptides Are Recognized by Human T Cells in the Context of DRB1*04:01. Diabetes, 69(7), 1492-1502.

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Hematopoietic Lineage Analysis in Knockout Mice

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For analysis of hematopoietic lineage markers in the BMs of mice with specific knockout of Wls or Six1 in hematopoietic cells, we follow protocols described previously [30 (link)]. Characterization of MLL-AF9 AML and isolation of the enriched LIC population from BMs of AML mice have been described previously [28 (link)]. Fluorescein-labeled antibodies used in the study include: Biotin Mouse Lineage Depletion Cocktail (BD Biosciences); Streptavidin-PerCP-Cy5.5, Sca1-FITC, cKit-APC-Cy7, cKit-PE, FcγR-PE-Cy7, CD34-PacificBlue, FLT3-PE, IL7R-APC, Mac1-APC, B220-PE, CD3-APC, Gr1-PE, Mac1-APC, CD16/CD32, all antibodies are anti-mouse and were purchased from eBiosciences.

Zhang L.S., Kang X., Lu J., Zhang Y., Wu X., Wu G., Zheng J., Tuladhar R., Shi H., Wang Q., Morlock L., Yao H., Huang L.J., Maire P., Kim J., Williams N., Xu J., Chen C., Zhang C.C, & Lum L. (2018). Installation of a cancer promoting WNT/SIX1 signaling axis by the oncofusion protein MLL-AF9. EBioMedicine, 39, 145-158.

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T Cell Activation Assay with DCs

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The T cells were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma–Aldrich, USA) according to the manufacturer’s instructions, and then cocultured with DC, Rapa-DC or CsA-DC. After 5 days of coculture, the cells were harvested and labelled with the following fluorochrome-conjugated monoclonal antibodies: CD3-APC (clone: OKT3, eBioscience, USA), CD69-FITC (clone: FN50, eBioscience, USA) and CD25-PE (clone: BC96, BD Pharmingen, USA) to identify T cells or CD11c-APC and PD-L1-FITC (clone: MIH1, BD Pharmingen, USA) to identify DCs. All procedures were performed according to the manufacturers’ protocols. The cells were prepared for flow cytometry analysis and analysed as described above.

Machcińska M., Kotur M., Jankowska A., Maruszewska-Cheruiyot M., Łaski A., Kotkowska Z., Bocian K, & Korczak-Kowalska G. (2021). Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms. Archivum Immunologiae et Therapiae Experimentalis, 69(1), 27.

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Assessing Apoptosis in Cancer Cell Lines

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Activated T cells were co-cultured with SNU-449 or SK-Hep-1 at different E/T ratios for 24 h. The apoptosis percentage of SNU-449 and SK-Hep-1 cells was detected using flow cytometry using Annexin V-APC/PI Apoptosis Kit (KeyGEN BioTECH) according to the manufacturer’s instructions.
For flow cytometry, cells were washed in PBS and centrifuged at an appropriate speed. The cells were then fixed with 4% PFA, washed twice, and blocked with 5% BSA in PBS. After centrifugation, the cells were incubated in the primary antibodies for 30 min. The cells were then washed again and analyzed using flow cytometry (Becton Dickinson). For each independent sample, a total of 10,000 cells were collected for analysis.
The primary antibodies were as follows: GzmB-PE (372208; BioLegend), PD-L1-APC (17-5938-42; Invitrogen), perforin-APC (308112; BioLegend), CD62L-PC5.5 (45-0621-82; eBioscience), CD8-PE (12-0084-82; eBioscience), PD-1-PECy7 (25-9985-82; eBioscience), CD4-FITC (11-0041-82; eBioscience), CD3-APC (17-0031-82; eBioscience), CD8-PEcp-cy5.5 (45-0088-42; eBioscience), and CD44-PECy7 (25-0441-82; eBioscience).

Zhao B., Zheng X., Wang Y., Cheng N., Zhong Y., Zhou Y., Huang J., Wang F., Qi X., Zhuang Q., Wang Y, & Liu X. (2024). Lnc-CCNH-8 promotes immune escape by up-regulating PD-L1 in hepatocellular carcinoma. Molecular Therapy. Nucleic Acids, 35(1), 102125.

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Multiparametric Immune Cell Analysis

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All antibodies were purchased from commercial sources: CD3-phycoerythrin (PE), CD4-allophycocyanin (APC), CD4-peridinin-chlorophyll–protein complex (PerCP), CD19-PE, CD3-APC, CD3-fluorescein isothiocyanate (FITC), CD8-PE, NK1.1-APC, F4/80-PE, CD11c-PE, CD11c-APC, IL-4-PE, IFN-γ-APC, TNFα-FITC, CD69-APC, anti-CD3 Abs, and anti-CD28 Abs from eBioscience; Rabbit polyclonal GIT2 Abs, anti-phospho-PAK1/2 (Ser199/204 of PAK1 and Ser192/197 of PAK2 Abs, and anti-phospho-c-Cbl (Y774) Abs from Cell Signaling; Anti-phospho-Erk1/2 (E4) Abs, anti-PAKα, anti-Erk1/2 (K23) from Santa Cruz.

Hao Y.E., He D.F., Yin R.H., Chen H., Wang J., Wang S.X., Zhan Y.Q., Ge C.H., Li C.Y., Yu M, & Yang X.M. (2015). GIT2 deficiency attenuates concanavalin A-induced hepatitis in mice. FEBS Open Bio, 5, 688-704.

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