Cd3 ecd

Manufactured by Beckman Coulter

Sourced in United States

The CD3-ECD is a fluorescent-labeled monoclonal antibody used for the identification and enumeration of T cells in flow cytometry analysis. It binds to the CD3 complex, which is expressed on the surface of mature T cells. The CD3-ECD antibody is conjugated with the Phycoerythrin-Texas Red (ECD) fluorochrome, enabling the detection and quantification of CD3-positive cells.

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26 protocols using cd3 ecd

Flow Cytometry Profiling of Lymphocyte Subsets

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Flow cytometry analysis was performed to quantify lymphocyte subsets on BD LSR II flow cytometer with BD FACS DIVA software (BD Biosciences, Heidelberg, Germany). The following antibodies were used for phenotypic analysis: CD45-FITC, CD19/20-PE, CD14-Cy-7, CD25-FITC, CD127-PE, CD45RA-APC, CD3-APC Cy-7, CD8-FITC, CD4-PE, CD4-APC Cy7, CD8-APC Cy7, CD62L-FITC, CCR7-PE, CD27-FITC, CD62L-PE, (Beckton Dickinson, Franklin Lakes, NJ, USA), CD3-ECD, CD56-APC, CD45RO-ECD (Beckman Coulter, Miami, FL, USA).^{30 (link)}

Triplett B.M., Shook D.R., Eldridge P., Li Y., Kang G., Dallas M., Hartford C., Srinivasan A., Chan W.K., Suwannasaen D., Inaba H., Pui C.H, & Leung W. (2015). Rapid Memory T-cell Reconstitution Recapitulating CD45RA-depleted Haploidentical Transplant Graft Content in Patients with Hematologic Malignancies. Bone marrow transplantation, 50(7), 968-977.

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Multicolor Flow Cytometry Panel

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The following Abs were used in different combinations. CD8-PB, CD8-APCH7, CD3-APCH7, CD45RA-PECy5, PD-1-FITC, IFN-γ-APC, TNF-α-PECy7, and IL-2-PE were purchased from Becton Dickinson (BD, San Diego, CA), CD4-ECD, CD3-ECD, CD28-ECD, CD27-APC from Beckman Coulter (Fullerton, CA, USA), perforin-FITC (clone B-D48) from Diaclone (Besançon, France), CCR7-FITC from R&D Systems (Minneapolis, MN, USA), 2B4-PE-Cy5.5 and CD160-APC from Biolegend (San Diego, CA, USA) and CD127-PE-Cy7 from eBioscience (San Diego, CA, USA).

Romano E., Michielin O., Voelter V., Laurent J., Bichat H., Stravodimou A., Romero P., Speiser D.E., Triebel F., Leyvraz S, & Harari A. (2014). MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial. Journal of Translational Medicine, 12, 97.

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Multiparametric Flow Cytometry Analysis

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Acquisitions were performed using a Beckman Coulter Cytoflex flow cytometer, and data were analyzed by the Kaluza software (Beckman Coulter, Milan, Italy). Cytoflex Daily QC Fluorospheres (Beckman Coulter) were used to calibrate the flow cytometer. The following monoclonal fluorochrome-conjugated antibodies were used for cell labeling of CD14⁺ cells from NKEV-monocyte cultures and PBMCs harvested from LPS-stimulated NKEV-PBMC cocultures: CD3-KO (Beckman Coulter), CD14-Alexa 750 (Beckman Coulter), CD16-BV650 (Biolegend), CD80-86-PE (Becton Dickinson), and HLA-DR-APC (Beckman Coulter).
The following antibodies were used for cell labeling of peripheral blood lymphocytes (PBL), NK cells and PBMCs harvested from CD3/CD28-stimulated or unstimulated NKEV-PBMC cocultures, in the presence or absence of TGFβ/IL-10: CFSE (CellTrace, Thermo Fisher Scientific), CD3-ECD (Beckman Coulter), CD4-Alexa700 (Beckman Coulter), CD8-BV605 (Biolegend), CD56-BV510 (Becton Dickinson), CD16-BV650 (Biolegend), PD-1-PC7 (Beckman Coulter), CD25-PercpCy5.5 (Becton Dickinson), HLA-DR-APC (Beckman Coulter), and CD14-Alexa750 (Beckman Coulter).
Samples were incubated with Fc blocking reagent (Miltenyi Biotec, Germany) for 10 min at room temperature before the addition of monoclonal antibodies for 40 min at 4°C. Thereafter, samples were washed and fixed.

Federici C., Shahaj E., Cecchetti S., Camerini S., Casella M., Iessi E., Camisaschi C., Paolino G., Calvieri S., Ferro S., Cova A., Squarcina P., Bertuccini L., Iosi F., Huber V, & Lugini L. (2020). Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors. Frontiers in Immunology, 11, 262.

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Flow Cytometric Analysis of NK Cell Receptors

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The antibodies used in this study were CD56 PE-Cy7 and APC-Cy7, CD158a/CD158b/CD158e1 PE (used in experiments were KIR were pooled), CD158e1 BV421, TNF-α AF647, IFN-γ Pacific blue, DLL1 and DLL4 PE, purified mouse IgM isotype control (BioLegend), CD158b FITC, CD107a FITC, purified mouse anti-human CD16 (BD Bioscience), CD3 ECD, CD158b APC (Beckman Coulter), and CD158a PE-Cy7 (eBioscience). For CD16 activation studies, cells were cultured with anti-CD16 or isotype control for 30 min and then crosslinked with goat-anti mouse IgG for 5 hours. Staining, acquisition, and analysis were performed as previously described (27 (link)). Finally, cells were run on an LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (TreeStar).

Felices M., Ankarlo D.E., Lenvik T.R., Nelson H.H., Blazar B.R., Verneris M.R, & Miller J.S. (2014). Notch signaling at later stages of Natural Killer cell development enhances KIR expression and functional maturation. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3344-3354.

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Flow Cytometric Analysis of Cytokine Production

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PBMC were incubated with PPD, ESAT-6, PHA or medium alone at 37°C in 5% CO₂ for 72 hours. Brefeldin A (eBioscience, BFA, final working concentration 3uL/mL) was added to the cells 4 hours before staining. Antibodies were prepared, cells transferred to tubes then spun down at 750 g for 5 minutes at 4°C and washed once in 500 uL FACS buffer. Samples were resuspended in 100 uL of diluted antibody against human cell-surface markers (CD4-PerCP-Cy5.5 (eBioscience,USA), CD8-PE/CYTM7 (BD,USA), or CD3-ECD (Beckman Coulter, France)), incubated for 30 minutes at 4°C, and washed twice in 500 uL FACS buffer. Supernatants were removed and cells were resuspended in 100 uL of Cytofix/Cytoperm(BD,USA), then incubated 20 minutes at 4°C. After washing cells, cells were suspended in diluted intracellular antibodies(PE-IL-17 (BD,USA), Alexa Fluor-IFN-γ (BD,USA), PE-isotype (BD,USA) or Alexa Fluor-isotype (BD,USA)), incubated 30 minutes at 4°C, then washed in 500uL 1X Perm Wash and resuspended in 100 uL of 1% paraformaldehyde. Data were collected using a BECKMAN COULTER Cytomics FC 500 Flow Cytometry System and analyzed with CXP software.

Fan L., Xiao H., Mai G., Su B., Ernst J, & Hu Z. (2015). Impaired M. tuberculosis Antigen-Specific IFN-γ Response without IL-17 Enhancement in Patients with Severe Cavitary Pulmonary Tuberculosis. PLoS ONE, 10(5), e0127087.

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NK Cell Immunophenotyping in Peripheral Blood

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All participants donated 10 ml of peripheral blood anticoagulated with EDTA, and samples were processed within three hours after extraction. Briefly, 150 μl of peripheral blood was incubated with CD56-Pacific blue (362520), CD16-BrilliantViolet510 (302048), CD19-PercPCy5.5 (302230), NKG2A-APC fyre (375116), NKG2C-PE (375004), NKG2D-FITC (320820) and NKp44-APC (325110) from Biolegend (San Diego, CA, USA) and CD3-ECD (A07748) from Beckman Coulter (Brea, CA, USA), according to manufacturer’s protocols. After monoclonal incubation, red blood cells were lysed with BD FACS lysing solution (349202, Becton Dickinson, Franklin Lakes, NJ, USA). Samples were washed and suspended in 300 μl phosphate saline buffer, and acquired in a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA). For every variable, all files were collected and data was analyzed at the same time with Kaluza v2.1 (Beckman Coulter, Brea, CA) by a blinded operator. NK^bright, NK^dim and NKT populations were gated by expression of CD56, CD16 and CD3. Subpopulations expressing NKG2A, NKG2C and NKG2D were selected, and NKp44 expression was analyzed in every subpopulation with two different gating strategies to confirm the differences. Gating strategies are detailed in Supplementary File.

Abarca-Zabalía J., González-Jiménez A., Calle-Rubio M., López-Pastor A.R., Fariña T., Ramos-Acosta C., Anguita E., Urcelay E, & Espino-Paisán L. (2023). Alterations in the immune system persist after one year of convalescence in severe COVID-19 patients. Frontiers in Immunology, 14, 1127352.

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Multiparametric Flow Cytometry Analysis

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Cells were phenotypically analyzed using a Navios™ instrument with 10-color PMTs and three solid-state lasers (Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using Kaluza™ software (Beckman Coulter). In order to guarantee that the optics, laser, fluidics and fluorescence intensity were stable during all measurements calibration was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor + Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA). Cells were washed with PBS with 1% bovine serum albumin before being labelled with fluorochrome-conjugated mAbs. After incubation for 30 min at 4°C in the dark, cells were washed twice to remove unbound antibodies and analyzed. For cell surface staining, the following mAbs were used: CD3-ECD (A07748), CD16-FITC (IM0814U), CD45-Krome Orange (A96416), CD56-APC-Alexa Fluor750 (custom made), CD158a-APC-Alexa Fluor700 (custom made), CD158b-PC7 (A66901), CD158e1/e2-APC (A60795), CD159a-PC5.5 (custom made) (all from Beckman Coulter, Marseille, France) and CD159c-PE (FAB138P; R&D).

Kleinnijenhuis J., Quintin J., Preijers F., Joosten L.A., Jacobs C., Xavier R.J., van der Meer J.W., van Crevel R, & Netea M.G. (2014). BCG-induced trained immunity in NK cells: role for non-specific protection to infection. Clinical immunology (Orlando, Fla.), 155(2), 213-219.

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Profiling pSTAT3 Expression in PTCL, NOS

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Flowcytometric assay was performed on three PTCL, NOS cases, using one excised tonsil case as control. All PTCL, NOS cases were diagnosed in our laboratory using a T-cell lymphoma panel recommended by Euroflow and excluded from PTCL of T follicular helper (Tfh) cell phenotype (19 (link)) on a Cytek Spectrum Flowcytometer (NL-CLC, V16-B14). The detection of pSTAT3-Y705 was by using commercially available antibody (Catalog No: 612569) from BD Pharmingen (BD Bioscience, USA) and pSTAT3-S727 by conjugating fluorescence tag (FlexAble Coralite V405 Antibod Labeling Kit For Rabbit IgG, Cat No: KFA006) with pSTAT3-S727 (ab32143, Abcam) from Abcam (Cambridge, UK), in an 8-color panel consisting of pSTAT3-Y705-FITC, pSTAT3-S727-Corolite V405, CD3-ECD (Catlog No: A07748, Beckman Coulter), CD8-PE-Cy7 (Catlog No: 6607102, Beckman Coulter), CD10-PE-Cy5 (Catlog No: A07761, Beckman Coulter), CD19-PE-Cy5.5 (Catlog No: A66328, Beckman Coulter), CD45-cFlourV547 (Catlog No: R7-10011, Cytek), CD4-cFlourV610 (Catlog No: R7-20073, Cytek) and PD-1-BV750 (Catlog No: 329966, Biolegend).

Xiang C., Wu W., Fan M., Wang Z., Feng X., Liu C., Liu J., Liu G., Xia L., Si H., Gu Y., Liu N., Luo D., Wang Y., Ma D., Hu S, & Liu H. (2023). Phosphorylated STAT3 as a potential diagnostic and predictive biomarker in ALK- ALCL vs. CD30high PTCL, NOS. Frontiers in Immunology, 14, 1132834.

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Evaluating AML Therapy in NOD/SCID Mice

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NOD/SCID mice were bred and housed at the UHN/Princess Margaret Hospital (PMH; Toronto, Ontario, Canada) animal facility, and all studies were performed in accordance with guidelines approved by the UHN/PMH Animal Care Committee. Eight- to 12-week-old female NOD/SCID mice (10 per cohort) were sublethally irradiated (275 cGy) and interperitoneally injected with anti-CD122 antibody the day before intrafemoral transplantation. Freshly thawed primary AML samples harvested from patients’ peripheral blood were transplanted at cell doses of 5e6/mouse. At day 21, post transplantation, AGS67E and an isotype control ADC were dosed by i.v. injection at 1.5 mg/kg, QW for a total of 4 doses. Mice were sacrificed 7 days after the last treatment to assess the efficacy of AGS67E determined by the human AML engraftment in the injected right femur and non-injected bone marrow (left femur and two tibias). AML outgrowth was evaluated by flow cytometry using the following antibodies: CD45-FITC (BD), CD33-APC (BD), CD34-PE-Cy5 (Beckman Coulter), CD3-ECD (Beckman Coulter), CD38-PE-Cy7 (BD), and AGS67C-Biotin. Secondary detection of biotinylated antibodies utilized streptavidin–PE. Samples were analyzed using an LSRII flow cytometer (BD).

Pereira D.S., Guevara C.I., Jin L., Mbong N., Verlinsky A., Hsu S.J., Aviña H., Karki S., Abad J.D., Yang P., Moon S.J., Malik F., Choi M.Y., An Z., Morrison K., Challita-Eid P.M., Doñate F., Joseph I.B., Kipps T.J., Dick J.E, & Stover D.R. (2015). AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody–Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Molecular cancer therapeutics, 14(7), 1650-1660.

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Quantifying AML Myeloid Blast and LSC Populations

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PBMCs from AML patients were isolated and incubated with a cocktail of CD45-FITC (BD), CD33-APC (BD), CD34-PE-Cy5 (Beckman Coulter), CD3-ECD (Beck-man Coulter), CD38-PE-Cy7 (BD), and either AGS67C-Biotin or isotype-Biotin antibodies. Secondary detection for biotinylated antibodies was accomplished with Streptavidin-PE. An LSRII flow cytometer (BD) was used for acquisition. Within the CD45⁺ population, two distinct subpopulations were defined, CD33⁺/CD3⁻/CD20⁻ (myeloid blasts) and CD33⁺/CD3⁻/CD34⁺/CD38⁻ [leukemic stem cells (LSC)]. Analysis was done with FlowJo version 9.5.4. MFIR for each AML sample was calculated by dividing the AGS67C MFI by the isotype MFI.

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