Cd3 fitc clone ucht1

Manufactured by BD

Sourced in United States

CD3-FITC (clone UCHT1) is a fluorescently labeled antibody targeting the CD3 molecule on the surface of T cells. It is commonly used in flow cytometry applications to identify and quantify T cell populations.

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7 protocols using cd3 fitc clone ucht1

Multiparameter Flow Cytometry Analysis of BAL

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All patients underwent bronchoscopy in sedation or general anaesthesia. BAL was performed by instillation of 200 ml saline in the middle lobe. BAL samples were kept on ice, filtered through gauze (Gazin, Lohmann & Rauscher, Austria) and centrifuged at 400 × g for 15 min at 4 °C. Erythrocyte lysis was performed when appropriate.
For flow cytometry, BAL cells were treated with Fc Block (clone Fc1.3216, BD Pharmingen, CA, USA) prior surface marker staining and DAPI (Invitrogen, Thermo Fisher scientific, CA, USA) for dead cell exclusion. Antibodies used were anti-human CD45-BV510 (clone HI30, BD Horizon, CA, USA), CD3-FITC (clone UCHT1, BD Pharmingen, CA, USA), CD4-PE-Cy7 (clone SK3, BD Pharmingen, CA, USA), CD8-ECD (clone SFCI21Thy2D3, Beckman Coulter, CA, USA) and CD279-APC (PD-1) (clone MIH4, BD Pharmingen, CA, USA). Flow cytometric analysis was conducted using a CytoFLEX V5-B5-R3 Instrument (Beckman Coulter, CA, USA) equipped with 405 nm, 488 nm and 638 nm lasers running with CytExpert software v2. Data analysis was performed using FlowJo v10 (LLC Software, SD, USA).
In patients with pulmonary lesions suspicious for lung cancer, transbronchial biopsies or endobronchial ultrasound-guided transbronchial needle aspiration were performed in the same procedure to obtain histological diagnosis in the majority of patients.

Hammer B., Bal C., Gysan M., Zehetmayer S., Geleff S., Zech A., Kramer M., Ayazseven S., Idzko M., Mosleh B., Hoda M.A, & Gompelmann D. (2023). Evaluation of PD-1 T lymphocytes in bronchoalveolar lavage in lung cancer compared to benign lung diseases. Clinical and Experimental Medicine, 23(8), 5177-5182.

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Multiparametric Flow Cytometry of Blood, Spleen, and Tumor

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Whole blood, spleen, and tumor tissues were processed for flow cytometry analysis. In brief, gentleMACS Octo Dissociator from Miltenyi Biotec was used for spleen and tumor-tissue dissociation, followed by filtering through 100 µm screens. After tissue dissociation, the cells were washed with PBS and incubated with the antibody panel for 15–20 min at room temperature. The red blood cells were lysed by Pharm Lyse (BD Biosciences, San Jose, CA, USA) for 8–12 min following antibody staining at room temperature. Live or dead cells were detected by LIVE/DEAD Fixable Red Dead Cell Stain Kit (Thermo Fisher Scientific). Samples were centrifuged and washed with PBS before suspension in PBS for data acquisition on BD FACSCanto II using FACSDiva software (BD Biosciences).
Fluorochrome–conjugated mAb to the following human antigens were used: CD45-V510 (clone HI30), CD45-phycoerythrin (PE; clone HI30), CD3-FITC (clone UCHT1), CD4-PE-cyanine 7 (clone SK3), CD8-allophycocyanin-cyanine 7 (clone SK1), CD19-allophycocyanin (clone HIB19), PD-1-PE (clone EH12.2H7), and PD-L1-V421 (clone 29E.2A3). All antibodies were purchased from BioLegend.

Wang M., Yao L.C., Cheng M., Cai D., Martinek J., Pan C.X., Shi W., Ma A.H., De Vere White R.W., Airhart S., Liu E.T., Banchereau J., Brehm M.A., Greiner D.L., Shultz L.D., Palucka K, & Keck J.G. (2017). Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. The FASEB Journal, 32(3), 1537-1549.

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Multiparametric Flow Cytometry for HLA-G+ CD4+ T Cells

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Flow cytometry was performed on BDFACS Canto II Flow Cytometer (BD Biosciences, USA) and data analysis was performed on FlowJo v10. Cell sorting was performed using BDFACSAria III (BD Biosciences) by gating on single viable HLA-G⁺CD4⁺ and HLA-G⁻CD4⁺ cells reaching a purity of sorted cell populations over 95%. The following monoclonal anti-human antibodies were used: HLA-G-PE (clone MEM- G9, Sigma-Aldrich), CD4 FITC/PerCP/APC-Cy7 (clone RPA-T4, BD Biosciences or BioLegend) and CD3-FITC (clone UCHT-1, BD Biosciences), IDO-1-Alexa Fluor 647 (clone V50-1886, BD Biosciences), CD45RA-PE-Cy7 (clone HI100, BioLegend), CD62L-V450 (clone DREG-56, BD Biosciences), CD25-PE (clone M-A251, BioLegend), CD127-PerCP-Cy5.5 (clone HIL-7R-M21, BD Biosciences), and GITR-BV421 (clone V27-580, BD Biosciences). Dead cell exclusion was performed with Zombie Aqua/NIR Fixable viability dye (BioLegend) or Far-Red Live/Dead Fixable dead cell stain kit (Invitrogen, USA). Fixation was performed using BD Phosflow Fix Buffer (BD Biosciences), and permeabilization using perm buffer (PBS, Triton 0.5%, and BSA 0.5%). Fluorescence minus one (FMO) control was used to set the threshold of positivity for HLA-G, CD45RA, CD62L, CD25, CD127, and GITR.

Lysandrou M., Stamou P., Kefala D., Pierides C., Kyriakou M., Savvopoulos N., Christofi P., Papadopoulou A., Yannaki E., Costeas P, & Spyridonidis A. (2023). Hypomethylation-induced regulatory programs in T cells unveiled by transcriptomic analyses. Frontiers in Immunology, 14, 1235661.

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PBMC Isolation, Sorting, and RNA-Seq

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Cryopreserved PBMC were thawed in media containing 20% fetal bovine serum. Cell counts and viabilities were assessed using Guava ViaCount reagent and a Guava PCA (Millipore Sigma). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes), washed, and blocked using normal mouse IgG (Caltag). The cells were surface stained for CD3 FITC (clone UCHT1, Becton Dickinson [BD]), CD8 PerCP-eF710 (SK1, eBiosciences), CD14 V500 (M5E2, BD), CD45RO eF650NC (UCHL1, eBiosciences), CD4 APC (RPAT4, BD), CD45RA APC-H7 (HI100, BD), CD19 PE-Cy5 (SJ25-C1, Invitrogen), and CD56 PE-Cy7 (NCAM16.2, BD) (Figure S2). The cells were then washed and sorted/analyzed on the BD FACSAria II SORP Cell Sorter. Total RNA from sorted cell subsets was extracted using the Single Cell RNA Purification kit (Norgen Biotek Corp). RNA was prepared for NGS RNA sequencing (RNAseq) using the SMART-Seq technology as described previously (Ehrenberg et al., 2019 ; Picelli et al., 2014 (link); Ramsköld et al., 2012 (link); Shangguan et al., 2021 ). Briefly, cDNA was generated per manufacturer’s instructions from 2.5 ng of RNA, final exogenous ERCC RNA spike-in dilutions of 1:190,000, and 11 cycles of library PCR amplification. A total of 300 ng of cDNA was input template for final library processing using the Nextera XT kit (Illumina) per manufacturer’s instructions.

Li S.S., Hickey A., Shangguan S., Ehrenberg P.K., Geretz A., Butler L., Kundu G., Apps R., Creegan M., Clifford R.J., Pinyakorn S., Eller L.A., Luechai P., Gilbert P.B., Holtz T.H., Chitwarakorn A., Sacdalan C., Kroon E., Phanuphak N., de Souza M., Ananworanich J., O’Connell R.J., Robb M.L., Michael N.L., Vasan S, & Thomas R. (2022). HLA-B*46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype. Cell host & microbe, 30(8), 1173-1185.e8.

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Multiparametric Flow Cytometry Immunophenotyping

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The following anti-human antigen antibodies were used for flow cytometry: CD3-FITC (clone UCHT1), CD3-APC (clone UCHT1), CD4-FITC (clone RPA-T4), CD8-APC (clone RPA-T8), CD16-PE (clone 3G8), CD25-PE (clone BC96), CD28-FITC (clone CD28.2), CD66b-FITC (clone G10F5), CD69-PE (clone FN50), CD279-FITC (clone MIH4), Vβ-3-PE (clone KJ25) from BD Biosciences (San Jose, CA, USA); CD152-APC (clone L3D10) from BioLegend (San Diego, CA, USA); LAG-3-PerCP (#FAB2319C) and TIM-3-AlexaFluor488 (clone 344823) from R&D Systems (Minneapolis, MN, USA); CD57-PE-Cy7 (clone TB01) from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA), TIGIT-PE (clone MBSA43) from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). For flow cytometry analysis, 0.2 × 10⁶ cells were pelleted, 1 µl of the antibody was added and the tube was vortexed shortly. After 15 min of incubation in the dark, cells were washed with 1 ml PBS and resuspended in 200 µl PBS/1% PFA. Analysis was done at BD FACS Canto™ II (BD Biosciences, San Jose, CA, USA).

Vonwirth V., Bülbül Y., Werner A., Echchannaoui H., Windschmitt J., Habermeier A., Ioannidis S., Shin N., Conradi R., Bros M., Tenzer S., Theobald M., Closs E.I, & Munder M. (2021). Inhibition of Arginase 1 Liberates Potent T Cell Immunostimulatory Activity of Human Neutrophil Granulocytes. Frontiers in Immunology, 11, 617699.

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Flow Cytometric Analysis of Foxp3+ Regulatory T Cells

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Cells were washed with PBS and incubated with antibodies diluted in PBS/10% HS/0.01% NaN3 (sodium azide) for 30 min on ice. Surface antibodies were anti-CD4 PerCp (clone SK3), CD3 FITC (clone UCHT1), and CD25 PE (clone M-A251, all BD Biosciences). Intracellular staining with anti-Foxp3 APC (clone PCH101, eBiosciences) was performed using the eBioscience fixation/permeabilization buffer kit. A minimum of 10⁵ events in the lymphocyte gate was acquired using a FACScalibur flow cytometer for 4-color analysis and analyzed using WEASEL software (WEHI, Melbourne, VIC, Australia). Cells were gated first based on forward and side scatter to excluded dead cells and cell debris. T cells in the lymphocyte gate were identified based on CD3 expression, further sub-gated on CD4+ T cells (Figure 1A) and CD25+ cells then subdivided into Foxp3hi and Foxp3int cells (Figure 1B).

Scholzen A., Cooke B.M, & Plebanski M. (2014). Plasmodium falciparum induces Foxp3hi CD4 T cells independent of surface PfEMP1 expression via small soluble parasite components. Frontiers in Microbiology, 5, 200.

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Flow Cytometric Immunophenotyping of Cells

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The single cells were suspended in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin as buffer. The human and mouse F_C receptors were blocked by human BD Fc block and mouse BD Fc block, respectively. The cell suspension was incubated with fluorochrome-conjugated monoclonal antibodies in dark, at room temperature, for 30 min. Cells were washed twice with buffer afterward, and then proceeded analysis on a Beckman Coulter Gallios flow cytometer. The dead cells were excluded by using 7-amino-actinonycin D (7-AAD). The data were analyzed on FlowJo software. The appropriate isotype antibodies were used as controls. The fluorochrome-conjugated monoclonal antibodies were used as follows; for human: CD138-BV421 (clone MI15, Biolegend), CD38-FITC (clone HB-7, BD), CD29-APC (clone TS2/16, Biolegend), CD49d-PE-CF594 (clone 9F10, BD), CD3-FITC (clone UCHT1, BD), CD19-BV421 (clone HIB19, BD), CD14-PE-CF594 (cloneMOP9, BD), CD45-BV510 (clone HI30, BD) and for mouse: CD138-BV421 (clone 281–2, BD), CD29-APC-eFluor780 (clone HMb1-1, eBioscience), CD49d-APC (clone R1-2, Biolegend), CD45-BV510 (clone 30-F11, Biolegend).

Hathi D., Chanswangphuwana C., Cho N., Fontana F., Maji D., Ritchey J., O’Neal J., Ghai A., Duncan K., Akers W.J., Fiala M., Vij R., DiPersio J.F., Rettig M, & Shokeen M. (2022). Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival. Scientific Reports, 12, 30.

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