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Anti cd3 antibody

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The Anti-CD3 antibody is a laboratory reagent used for the detection and isolation of T cells. It binds to the CD3 complex on the surface of T cells, allowing for their identification and separation from other cell types. The Anti-CD3 antibody is a useful tool in immunological research and cell biology applications.

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Spelling variants of this product

Anti-CD3 (541 citations) Anti-mouse CD3 antibody (10 citations) Anti-CD3/anti-CD28 antibodies (9 citations) FITC-conjugated anti-CD3 antibody (4 citations) APC-conjugated anti-human CD3 antibody (3 citations) FITC-labeled anti-CD3 antibody (3 citations) Anti-CD3-FITC antibody (3 citations) Human anti-CD3 antibody (clone SK7, (3 citations) APC anti-human CD3+ monoclonal antibody (2 citations) Anti-CD3 monoclonal antibody (mAb) (2 citations)

82 protocols using anti cd3 antibody

1

CFDA-SE Labeled PBMC Stimulation Assay

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PBMCs were isolated by centrifugation on Histopaque™ gradient (Sigma Chemical Co., USA) from venous peripheral blood collected in tubes containing EDTA as the anti-coagulant. Cells were then stained with 3 µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Sigma Chemical Co., USA), stimulated with 250 ng per 2 million cells of immobilized (tissue-culture plate-bound) anti-CD3 antibody (BD-Pharmigen, USA) and incubated for up to 5 days at 37 °C, 5% CO2 as previously described11 (link).
Stimulated cells were collected after 72 and 120 hours, stained with following antibodies: RPE-Cy5-conjugated anti-CD4 or anti-CD8 and PE-conjugated anti-CD28 (BD Pharmingen, USA), and analyzed with flow cytometry using FACScan instrument.
Lisowska K.A., Pindel M., Pietruczuk K., Kuźmiuk-Glembin I., Storoniak H., Dębska-Ślizień A, & Witkowski J.M. (2019). The influence of a single hemodialysis procedure on human T lymphocytes. Scientific Reports, 9, 5041.
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2

CFSE-Based T Cell Proliferation Assay

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PBMC were labeled with CFSE dye (Invitrogen) as described [30 (link)]. Briefly, cells were incubated with 5μM CFSE (in PBS containing 5% fetal bovine serum (FBS)) at 37°C for 5 min and washed three times with ice-cold PBS-5% FBS. 2x105 CFSE-labeled PBMCs/well of a 96-well plate were cultured in Roswell Park Memorial Institute (RPMI)1640 media supplemented with 10% FBS, 1% penicillin/streptomycin, 1mM Sodium pyruvate and 0.05mM 2-Mercaptoethanol in the presence of decreasing doses of anti-CD3 antibody (ref 555337, BD Pharmigen). PBMC were harvested after 2 and 5 days of culture and stained with anti-CD4-APC and anti-CD8-PE antibodies (eBiosciences) for subsequent analysis by flow cytometry (Fortessa, Becton Dickinson). Percentages of proliferating cells were analyzed by FlowJo software.
Carreras-Sureda A., Rubio-Moscardo F., Olvera A., Argilaguet J., Kiefer K., Mothe B., Meyerhans A., Brander C, & Vicente R. (2016). Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21. PLoS ONE, 11(11), e0166414.
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3

Quantifying NKT Cells and Cytokines

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All samples from individual subjects were tested simultaneously on blinded samples. For NKT measurements, ten ml of heparinized peripheral blood were collected before and 24 hours following each treatment, and at day 56 (end of study). PBMCs were isolated by Ficoll density gradient centrifugation and cryopreserved in liquid nitrogen until use. For staining, 1×106 PBMCs were incubated with α-GalCer or vehicle loaded CD1d-tetramers (ProImmune, UK) for 30 min at 4°C, then with anti-CD3 antibody (Becton–Dickinson Biosciences) for 30 min at 4 °C. Analysis was performed on a FACSCanto using CELLQuest software (BD Biosciences).
Cytokine was measured before, 4 and 24h after the first and second injections and at the end of study using a multiplex electrochemiluminescense assay according to the manufacturer’s instructions (MSD) and analyzed using the Discovery Workbench MSD software.
Tefit J.N., Crabé S., Orlandini B., Nell H., Bendelac A., Deng S., Savage P.B., Teyton L, & Serra V. (2014). Efficacy of ABX196, a new NKT agonist, in prophylactic human vaccination. Vaccine, 32(46), 6138-6145.
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4

Activating and Profiling CD4+ T Cells

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CD4+ T lymphocytes were isolated from frozen PBMC using the EasySep™ Human CD4+ T Cell Isolation Kit (Stem Cell Technologies, Vancouver, BC, Canada). CD4+ cells were resuspended at a concentration of 1 × 106 cells/mL in X-VIVO 20 serum-free medium (Lonza Group Ltd., Basel, Switzerland) supplemented with 1% penicillin/streptomycin. CD4+ cells (500,000 cells/well) were incubated in 48-well plates previously coated with an anti-CD3 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) with or without a CD28 antibody (Becton Dickinson, Franklin Lakes, NJ, USA), which induces cell activation. After 6 h of incubation at 37 °C with 5% CO2, the supernatant and cell pellets were collected for further analysis. The cytokines IL-17, IL-22, TNF-α, TFG-β, and IL-1β were measured in the culture supernatant by DuoSet® ELISA Kits (R&D Systems, Minneapolis, MN, USA). For the IL-13 measurement, a human IL-13 ELISA kit was used (Invitrogen, Thermo Fisher, USA). IL-21 interleukin was measured in the cell pellet by western blot.
Fiorillo A., Gallego J.J., Casanova-Ferrer F., Giménez-Garzó C., Urios A., Ballester M.P., Durbán L., Rios M.P., Megías J., San Miguel T., Kosenko E., Escudero-García D., Benlloch S., Felipo V, & Montoliu C. (2023). Mild Cognitive Impairment Is Associated with Enhanced Activation of Th17 Lymphocytes in Non-Alcoholic Fatty Liver Disease. International Journal of Molecular Sciences, 24(12), 10407.
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5

Blocking PD-1/PD-L1 Pathway Enhances Lymphocyte Proliferation

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Lymphocytes and DCs separated from cervical tissues were labeled with anti-CD3 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) and anti-CD11c antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Lymphocytes (CD3+) and DCs (CD11c+) were separated using MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). The density of cells was adjusted to 1 × 106 cells/mL using RPMI-1640 medium. Then, 100 μL cell suspension was added into 96-well culture plates (1 × 105 cells/well) for cultivation of 72 h. To block PD-1/PD-L1 pathway, anti-PD-L1 antibody (final concentration of 2 μg/mL) was added before incubation for 72 h. The cells were divided into normal control group, control + anti-PD-L1 antibody group, CIN group, CIN + anti-PD-L1 antibody group, cervical cancer group, and cervical cancer + anti-PD-L1 antibody group. Stimulator ConA was added into the medium (final concentration of 3 μg/mL), followed by incubation at 37°C in 5% CO2 for 72 h. MTT assay was performed to analyze lymphocyte proliferation. Culture supernatants from each group were subjected to CBA assay for the determination of IL-10 and TGF-β levels.
Chen Z., Pang N., Du R., Zhu Y., Fan L., Cai D., Ding Y, & Ding J. (2016). Elevated Expression of Programmed Death-1 and Programmed Death Ligand-1 Negatively Regulates Immune Response against Cervical Cancer Cells. Mediators of Inflammation, 2016, 6891482.
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6

Activation of Murine Myeloid Cells

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The following materials were commercially obtained from the sources indicated: Mitomycin C from Sigma–Aldrich (St. Louis, MO, USA); fetal bovine serum (FBS), mouse IFN-γ ELISA Ready Set Go!, mouse IL-2 ELISA Ready Set Go!, and an Alexa 546-conjugated anti-rabbit IgG antibody from Thermo Fisher Scientific (Waltham, MA, USA); Fc blocker (clone 2.4G2), an FITC-conjugated anti-CD11b antibody, an FITC-conjugated anti-Gr-1 antibody, an anti-CD3 antibody, and an anti-CD28 antibody from BD Biosciences (Franklin Lakes, NJ, USA); an anti-HDC antibody (ab37291) from Abcam (Cambridge, UK).
Kamei M., Otani Y., Hayashi H., Nakamura T., Yanai K., Furuta K, & Tanaka S. (2018). Suppression of IFN-γ Production in Murine Splenocytes by Histamine Receptor Antagonists. International Journal of Molecular Sciences, 19(12), 4083.
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7

Exosome Effects on CD4+ T Lymphocytes

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The effect of EphB2-Exos on CD4 + T lymphocytes in vitro was investigated by co-culturing CD4 + T lymphocytes and exosomes. A 96-well at-bottomed plate was pre-coated with anti-CD3 antibody (5 µg/mL, BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD28 antibody (10 µg/mL, BD Biosciences) overnight at 4°C to activate the CD4 + T lymphocytes. Subsequently, CD4 + T lymphocytes were added to the plate, which was washed twice with PBS. After 4 h, exosomes or EphB2-Exos were added, whereas CD4 + T lymphocytes were untreated (negative controls). The cells were maintained at 37°C for 3 days in a humidi ed incubator with 5% CO 2 . After 72 h of incubation, the CD4 + T lymphocytes were stained with PE-Cy7-anti-CD4 antibody (BD Biosciences), eFluor450-anti-IL-17A antibody (eBioscience, San Diego, CA, USA), PE-anti-Foxp3 antibody (eBioscience), or APC-anti-IFN-γ antibody (BD Biosciences). The stained cells were analyzed using ow cytometry.
Chu S., Yu T., Wang W., Wu H., Zhu F., Wei C., Gao F., Liu C, & Fan H. (2023). Exosomes derived from EphB2-overexpressing bone marrow mesenchymal stem cells regulate immune balance and repair barrier function. Biotechnology letters, 45(5-6).
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8

Cytokine Expression in Activated Murine Splenocytes

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Mouse splenocytes were isolated from eight-week-old mice. Six-well plates were coated with 1μg/ml of anti-CD3 antibody (BD, 553057) in sterile 1× PBS overnight at 4°C. Splenocytes were seeded in anti-CD3 antibody-coated six-well plates (4–6 × 106 cells per well) in RPMI 1640 medium containing 10% FBS, 1% glutamine, 50μM 2-ME, and 1% penicillin streptomycin. Splenocytes were then co-cultured with zymosan A (2μg/ml) for three days. After three days of culture, cells were harvested, and quantitative real-time RT-PCR was performed to measure the levels of various cytokines.
Zhu F., Willette-Brown J., Song N.Y., Lomada D., Song Y., Xue L., Gray Z., Zhao Z., Davis S.R., Sun Z., Zhang P., Wu X., Zhan Q., Richie E.R, & Hu Y. (2017). Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis. Cell host & microbe, 21(4), 478-493.e7.
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9

T-cell activation assay protocol

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96-well plates were incubated overnight at 37°C with 0.5 μg/ml anti-CD3 antibody (BD Pharmingen) in culture medium. Treg cells, CD4+ Th cells, or APCs were isolated as described in the previous section (Cell separations). 105 of each kind of cell suspended in culture medium were seeded into the wells of a 96-well plate. Samples in anti-CD3 coated wells were treated with anti-CD28 antibody (2.5 μg/ml) (BD Pharmingen), and incubated at 37°C, 5% CO2 for 4 days. Other samples were treated with APCs and 5 μg/ml of mycoplasma membrane antigen. These plates were incubated for 6 days, and supernatants were collected.
Odeh A.N, & Simecka J.W. (2016). Regulatory CD4+CD25+ T Cells Dampen Inflammatory Disease in Murine Mycoplasma Pneumonia and Promote IL-17 and IFN-γ Responses. PLoS ONE, 11(5), e0155648.
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10

Murine Anti-ITGB4 BiAb-Armed TDLN T Cells

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Murine anti-ITGB4 monoclonal antibody (Invitrogen) and anti-CD3 antibody (BD Biosciences, San Jose, CA) were coupled by Dr. Lawrence G. Lum Lab (University of Virginia) to produce murine anti-CD3/anti-ITGB4 BiAbs (mITGB4 BiAb). In order to induce 4T1 TDLN, 1 × 106 4T1 or 4T1-Luc tumor cells in 0.1 ml PBS were injected subcutaneously (s.c.) into the lower flanks of immunocompetent Balb/c mice. To induce SCC7 TDLN, 1 × 106 SCC7 tumor cells in 0.1 ml PBS were injected (s.c.) into the lower flanks of immunocompetent C3H mice. We used the same method to induce CT26 TDLN in Balb/c mice. Nine days after cells inoculation of the tumor cells, the TDLNs were harvested, and single-cell suspensions were prepared mechanically (38 (link)). TDLN cells were activated with anti-CD3 mAb and anti-CD28 mAb immobilized 6-well tissue culture plates for 2 days and expanded in CM containing 200 ng/ml of human rIL-2 (Pepro Tech Inc., Rocky Hill, NJ) for 3 days to generate activated/expended TDLN T cells. 1 × 106 activated TDLN T cells were incubated with 50 ng of mITGB4 BiAb for 1 h at 4 °C to generate murine ITGB4 BiAb-armed TDLN T cells (TDLN T-mITGB4 BiAb) (39 (link)).
Ruan S., Lin M., Zhu Y., Lum L.G., Thakur A., Jin R., Shao W., Zhang Y., Hu Y., Huang S., Hurt E.M., Chang A.E., Wicha M.S, & Li Q. (2019). Integrin β4-targeted cancer immunotherapies inhibit tumor growth and decrease metastasis. Cancer research, 80(4), 771-783.
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