Anti human cd3 antibody

Manufactured by BD

The anti-human CD3 antibody is a laboratory reagent used for the detection and quantification of CD3-positive T cells in various biological samples. It recognizes the CD3 complex, which is a key component of the T cell receptor. This antibody can be used in flow cytometry, immunohistochemistry, and other immunoassay applications to identify and analyze T cell populations.

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Anti-CD3 (541 citations) Anti-CD3 antibody (82 citations) Anti-human CD3 (37 citations) APC-conjugated anti-human CD3 antibody (3 citations) Mouse anti-human CD3 antibody (3 citations) Human anti-CD3 antibody (clone SK7, (3 citations) Purified mouse APC-conjugated anti-human CD3 antibody (2 citations) FITC-conjugated anti-human CD3 antibody (2 citations) APC‐H7‐conjugated anti‐human CD3 antibody (clone SK7; (2 citations) APC anti-human CD3+ monoclonal antibody (2 citations)

8 protocols using anti human cd3 antibody

Flow Cytometry Analysis of Meso-CAR Expression

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We used a FACSCalibur instrument and Cellquest software for all flow cytometry analysis. All cells in culture were washed with PBS before staining with antibodies. After 30 to 45 min of incubation at 4°C in the dark, cells were washed twice with PBS (Gibco, USA) and analyzed on the FACSCalibur instrument. Meso-CAR expression was monitored by flow cytometry using the Alexa Fluor 647-conjugated F(ab’)₂ antibody (Jackson Immunology, USA) directed against the scFv. Stained, un-transduced T cells or isotype antibodies were used as controls. Anti-human CD3, CD4, CD8, CD69, LAG-3, TIM-3, and PD-1 (CD279) antibodies were purchased from BD.
To measure T cells in mouse blood, 100 μL of venous blood was obtained weekly from the orbital venous plexus and stained with anti-human CD3 antibody. Flow cytometry data were analyzed using FlowJo software.

Sun Q., Zhou S., Zhao J., Deng C., Teng R., Zhao Y., Chen J., Dong J., Yin M., Bai Y., Deng H, & Wen J. (2018). Engineered T lymphocytes eliminate lung metastases in models of pancreatic cancer. Oncotarget, 9(17), 13694-13705.

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Adoptive Transfer of PBMC from CLL Patients

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PBMC from subjects with CLL (10⁸ cells/mouse) were adoptively transferred into NOD-scid IL2Rγ^null NSG mice [17 (link)] by intravenous injection. The mice were then treated with human GIFT4 protein (20 ng/mouse/day) or control cytokines for 6 days. On day 7, 100 μl of peripheral blood collected from each mouse were stained with anti-human CD3 antibody for 30 min, followed by addition of red blood cell lysis buffer (for 10 min) and AccuCheck Counting Beads (Invitrogen) to quantify circulating human T cell number as described [18 (link), 19 ]. On day 30, human T cells in the peripheral blood of NSG mice were also analyzed by FACS with anti-human CD3 antibody (BD) before the mice were sacrified. All mice used were female (6–8 weeks old) purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained in compliance with an IACUC protocol approved by Emory University.

Deng J., Pennati A., Cohen J.B., Wu Y., Ng S., Wu J.H., Flowers C.R, & Galipeau J. (2016). GIFT4 fusokine converts leukemic B cells into immune helper cells. Journal of Translational Medicine, 14, 106.

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Culturing cells from healthy and HCM patients

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Dermal fibroblasts from a healthy volunteer and a patient with HCM (case 1) obtained through a dermal biopsy were maintained in DMEM (Sigma) with 10% FBS (NICHIREI Biosciences Inc) and passaged twice. From a patient with HCM (case 2), peripheral blood mononuclear cells were separated via centrifugation of a heparinized whole blood sample, by using a Ficoll‐Paque PREMIUM (GE Healthcare) gradient. The mononuclear cells were seeded onto the anti‐human CD3 antibody (BD Pharmingen)‐coated 6‐well plates in 2 mL GT‐T502 medium (KOHJIN BIO) per well and incubated for 5 to 7 days until the activated T cells reached 80% to 90% confluency. Human iPSCs were maintained on irradiated MEF feeder cells in human iPSC culture medium: 80% DMEM/F12 (Sigma), 20% KO Serum Replacement (Invitrogen), 4 ng/mL basic fibroblast growth factor (bFGF; WAKO), 2 mmol/L l‐glutamine (Invitrogen), 0.1 mmol/L nonessential amino acids (Sigma), 0.1 mmol/L 2‐mercaptoethanol, 50 U/mL penicillin, and 50 mg/mL streptomycin (Invitrogen). The medium was changed every 2 days, and the cells were passaged by using 1 mg/mL collagenase IV (Invitrogen) every 5 to 7 days.

Tanaka A., Yuasa S., Mearini G., Egashira T., Seki T., Kodaira M., Kusumoto D., Kuroda Y., Okata S., Suzuki T., Inohara T., Arimura T., Makino S., Kimura K., Kimura A., Furukawa T., Carrier L., Node K, & Fukuda K. (2014). Endothelin‐1 Induces Myofibrillar Disarray and Contractile Vector Variability in Hypertrophic Cardiomyopathy–Induced Pluripotent Stem Cell–Derived Cardiomyocytes. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 3(6), e001263.

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Generating Human iPSCs from Blood Cells

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iPSCs were established as described previously [12] (link). Mononuclear cells were separated from the patient's blood by Ficoll–Paque PREMIUM (GE Healthcare). They were plated at 3.0 × 10⁴ cells/cm² in 6-well plates coated with anti-human CD3 antibody (BD Pharmingen). They were incubated at 37.0 °C, with 3.0% CO₂ conditions. Cells were infected with Sendai virus carrying Yamanaka factors (DINAVEC); transfectants were plated at 5.0 × 10⁵ in dish (MEF feeder) in the maintenance medium described previously [13] (link), [14] (link), and cultured for 4 weeks. ESC-like colonies were transferred to 24-well plates on mouse embryonic fibroblast feeders. TiPSCs and human ESCs were cultured following the method of culturing human ESCs described by Suemori et al. [13] (link).

Sogo T., Morikawa K., Kurata Y., Li P., Ichinose T., Yuasa S., Nozaki D., Miake J., Ninomiya H., Shimizu W., Fukuda K., Yamamoto K., Shirayoshi Y, & Hisatome I. (2016). Electrophysiological properties of iPS cell-derived cardiomyocytes from a patient with long QT syndrome type 1 harboring the novel mutation M437V of KCNQ1. Regenerative Therapy, 4, 9-17.

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T Cell Functional Avidity Assay

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Basically, the assay was performed using the same procedure with IFN-γ ELISpot assay as described above. PBMCs were cultured for 2 to 3 weeks in R10/50 after anti-human CD3 antibody (BD Pharmingen) stimulation, and culture media were changed from R10/50 to R10 two days before use. PBMCs were incubated with peptides at concentrations from 10^-5 to 10^-12 M, and SFU was calculated. The functional avidity to peptide dilutions was determined as a 50% of sigmoidal dose (SD₅₀) SFU.

Han C., Kawana-Tachikawa A., Shimizu A., Zhu D., Nakamura H., Adachi E., Kikuchi T., Koga M., Koibuchi T., Gao G.F., Sato Y., Yamagata A., Martin E., Fukai S., Brumme Z.L, & Iwamoto A. (2014). Switching and emergence of CTL epitopes in HIV-1 infection. Retrovirology, 11, 38.

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ELISPOT Assay for Cytokine Analysis

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Bacteria-stimulated moDCs were counted, washed with PBS, and cocultured with autologous PBL at a ratio of 1:20 in serum-free AIM-V medium for 3 days at 37 °C in a 5% CO₂ atmosphere. On day 4, the cells were collected, counted, and subjected to IFNγ, IL-17A, and IL-4 Ready Set Go ELISPOT assays according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Briefly, 300,000 cells/well were incubated in CTL-Test medium (Cellular Technology Limited, Cleveland, OH, USA) for 40–48 h at 37 °C in MultiScreen-HTS PVDF plates (Millipore S.A., Molsheim, France) precoated with capture antibodies specific for IFNγ, IL-4, or IL-17A. Together with the IL-4- and IL-17A-specific capture antibodies, 0.5 µg/mL purified antihuman CD3 antibody (BD Biosciences) was added to the coating buffer for the mitogenic stimulation of CD3⁺ T cells. The detection of the cytokine release was performed using biotinylated IFNγ-, IL-4-, or IL-17A-specific antibodies in the presence of HRP conjugated to avidin. Soon after, the colorigenic substrate, 3-amino-9-ethylcarbazole (AEC Substrate Set, BD Biosciences) was added. The color development was stopped by tap water, and air-dried plates were analyzed with a computer-assisted ELISPOT image analyzer (Series 1 ImmunoSpot Analyzer, ImmunoSpot Version 4.0 Software Academic, Cellular Technology Limited, Shaker Heights, OH, USA).

Tóth M., Muzsai S., Regulski K., Szendi-Szatmári T., Czimmerer Z., Rajnavölgyi É., Chapot-Chartier M.P, & Bácsi A. (2022). The Phagocytosis of Lacticaseibacillus casei and Its Immunomodulatory Properties on Human Monocyte-Derived Dendritic Cells Depend on the Expression of Lc-p75, a Bacterial Peptidoglycan Hydrolase. International Journal of Molecular Sciences, 23(14), 7620.

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CD70 Activation Assay with Jurkat Cells

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Nunc MaxiSorp 96-well plates (Thermo Fisher # 44-2404-21) were coated overnight at 4^oC with 1 μg/ml anti-human CD3 antibody (BD catalog # 555336) and dilutions of CD70 protein within 50 μl of PBS. The following day, plates were washed with PBS, and 50,000 Jurkat reporter cells were added in RPMI 1640 (Thermo Fisher # 72400047) with 10% FBS. The Jurkat reporter was stably transfected with an AP-1-dependent luciferase reporter. After a 4-h incubation at 37^oC, One-Glo luciferase reagent (Promega # E6120) was added to the cells, incubated with gentle shaking for 5 min, and luminescence was measured by a Tecan Spark plate reader.

Liu W., Maben Z., Wang C., Lindquist K.C., Li M., Rayannavar V., Lopez Armenta I., Nager A., Pascua E., Dominik P.K., Oyen D., Wang H., Roach R.C., Allan C.M., Mosyak L, & Chaparro-Riggers J. (2021). Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex. The Journal of Biological Chemistry, 297(4), 101102.

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Quantifying Treg Suppressive Capacity

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The suppressive capacity of Tregs was assessed by measuring inhibition of proliferation in mixed leukocyte reaction (MLR) assays. Proliferation was evaluated using the CellTrace^™ Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation kit (Invitrogen; Thermo Fisher Scientific, Inc.). Responder cells (human PBMCs) were labeled with 0.5 µM CFSE (Molecular Probes; Thermo Fisher Scientific, Inc.) prior to stimulation. CFSE-labeled responder cells (1×10⁵) from autologous Treg donors were stimulated with 2×10⁵ irradiated xenogeneic stimulator PBMCs or purified anti-human CD3 antibody (BD Biosciences). Tregs were titrated into the cultures at different ratios. After 7 days of culture, the proliferation of responder cells was analyzed by flow cytometry (LSR II; BD Biosciences).

Jin X., Hu M., Gong L., Li H., Wang Y., Ji M, & Li H. (2018). Adoptive transfer of xenoantigen-stimulated T cell receptor Vβ-restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice. Molecular Medicine Reports, 18(5), 4457-4467.

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