Anti human cd3 bv785

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Anti-human CD3-BV785 is a monoclonal antibody that binds to the CD3 complex on human T cells. It is conjugated to the fluorescent dye Brilliant Violet 785 (BV785) for use in flow cytometry applications.

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Anti-CD3 (468 citations) Anti-human CD3 (53 citations) Anti-CD3 BV785 (8 citations) CD3-BV785 (8 citations)

6 protocols using anti human cd3 bv785

Ectonucleotidase Expression on T-cell Subsets

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The expression of ectonucleotidases (CD39 and CD73) on αβDNT and activated Treg cells was evaluated by flow cytometry. The gating strategy for activated Treg cells was performed as previously described (5 (link)). PBMCs were stained with directly conjugated antibodies for 30 min at 4°C in the dark. The cells were washed before flow cytometry analysis. Antibodies used included anti-human CD3-BV785 (clone SK7), CD4-APC-fire750 (clone SK3), αβTCR-BV421 (clone IP26), CD56-BV510 (clone HCD56), CCR7 (CD197)-PE-cy7 (clone G043H7), HLA-DR-AF700 (clone L243), CD73-PE (clone AD2), CD39-BV605 (clone A1, BioLegend, San Diego, CA, USA), CD45RA-BV711 (clone HI100), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251), CD8-FITC (clone SK1), CD8-BUV395 (clone RPA-T8, BD Biosciences, San Diego, CA, USA), and the corresponding isotype controls. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Wang X., Zhang L., Du J., Wei Y., Wang D., Song C., Chen D., Li B., Jiang M., Zhang M., Zhao H, & Kong Y. (2022). Decreased CD73+ Double-Negative T Cells and Elevated Level of Soluble CD73 Correlated With and Predicted Poor Immune Reconstitution in HIV-Infected Patients After Antiretroviral Therapy. Frontiers in Immunology, 13, 869286.

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Isolation and Characterization of CAR-T Cells

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Isolation of immune cells from fresh blood was conducted by adding 1 mL ACK lysis buffer per 100 μL of blood, and lysis was performed on ice for 5 min. The lymphocyte fraction was depleted of red blood cells by the lysis. The cells were resuspended in the staining buffer (Thermo Fisher Scientific) and counted, then labeled with the viability stain (Zombie Aqua, BioLegend) in PBS for 15 min at room temperature, washed, and incubated with anti-human CD45-APC-Fire750 (Clone HI30, Biolegend) and anti-human CD3-BV785 (Clone UCHT-1, Biolegend) for 1 h at 4 °C in the staining buffer (Thermo Fisher Scientific). The cells were further washed and fixed in the intracellular (IC) fixation buffer (Thermo Fisher Scientific) overnight at 4 °C. Human CD45⁺ CD3⁺ CAR-T cells were analyzed using the Attune NxT flow cytometer.

Sugimoto H., Chen S., Minembe J.P., Chouitar J., He X., Wang H., Fang X, & Qian M.G. (2021). Insights on Droplet Digital PCR–Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy. The AAPS Journal, 23(2), 36.

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CFSE-based Lymphocyte Proliferation Assay

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To evaluate the proliferative responses of lymphocytes to C-Vx, carboxyfluorescein succinimidyl ester (CFSE) dilution method was used which is based on labelling cells with CFSE and evaluating the fluorescence halved by each cell division. PBMCs (up to 2 × 10⁷), freshly isolated and suspended in RPMI-1640 medium (Gibco, Paisley, UK), were stained with 1 μl of 5 mM CFSE solution (Thermo Fisher Scientific, USA) and incubated for 6 min at 4°C. Following washing with PBS, PBMCs were cultured for 120 h at 37°C with or without C-Vx (Miracle Labs PHArmaceutical Industry-Turkey) together with the absence or presence of 5 µl/mL PHA (Thermo Fisher, USA). Following cell culture, supernatants were collected and stored at −20°C for further analysis. PBMCs were harvested from the respective wells (US: unstimulated, PHA: phytohemagglutinin-stimulated, C-Vx, and PHA + C-Vx) into separate tubes for cell surface staining with anti-human-CD3-BV785, -CD4-PE-Cy7, -CD8-APC/Cy7, -CD16-BV570, and -CD56-BV711 (all from Biolegend, USA) mAbs. After the incubation, stained cells were washed with PBS and analyzed by flow cytometry.

Tahrali I., Akdeniz N., Yilmaz V., Kucuksezer U.C., Oktelik F.B., Ozdemir O., Cetin-Aktas E., Ogutmen Y., Ergen A., Abaci N., Tuzun E., Oncul O, & Deniz G. (2022). The modulatory action of C-Vx substance on the immune system in COVID-19. Emerging Microbes & Infections, 11(1), 2698-2710.

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Intracellular Cytokine Profiling of T Cells

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In order to determine the intracellular cytokine levels of T lymphocytes, PBMCs (1 × 10⁶ cells/mL) were pre-cultured with the presence or absence of C-Vx for 72 h and further stimulated with Cell Stimulation Cocktail [PMA (40.5 μM), Ionomycin (669.3 μM), and Brefeldin A (2.5 mg/mL)] (Biolegend, San Diego, USA) for 4 h at 37°C incubator. After the culture, cell surface staining was performed using anti-human-CD3-BV785, -CD4-PE, and -CD8-FITC monoclonal antibodies (mAbs) (all from Biolegend, San Diego, USA), prior to the detection of intracellular cytokine levels. Intracellular staining was performed using Fixation/Permeabilization Kit (BD Cytofix/Cytoperm, California, USA) according to the manufacturer’s protocol. Simply, cells were fixed and then permeabilized together with the addition of anti-IFN-γ-PE/Cy7, -TNF-α-APC/Cy7, -IL-4-APC, -IL-10-BV421, -IL-17-Alexa Fluor 700 mAbs. After washing, samples were measured by flow cytometry.

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CA9 DATEs Modulate T Cell Responses in ccRCC and GBM

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In ccRCC model, RCC243 and RCC243 CA9-KO cells were plated at 200,000 cells/well in 6 well plates the night prior to treatment. Human CD3⁺ T cells at an E:T ratio of 5:1 were added to the wells along with (1 nM) or without CA9 DATEs and incubated at 37°C in 5% CO₂ for 48 hours. The T cells were collected and stained for BV785 anti-human CD3 (BioLegend, Cat#317330), BV605 anti-human CD4 (BioLegend, Cat#317438), PE-anti-human CD8 (BioLegend, Cat#300908), and PE-CF594-anti-CD25 (BD Biosciences, Cat#562403) antibodies. Supernatants were collected and stored at -80°C for cytokine release analysis by enzyme-linked immunosorbent assay (ELISA).
In GBM models, GBM cells and T cells were co-incubated at a 1:1 ratio for 24 hours with (1μg = 13 nM) or without CA9 DATEs. The CD3⁺ (BD Pharmingen, Cat#563423) T cells and subpopulation of T cells including CD4⁺ (BD Pharmingen, Cat#555347) and CD8⁺ T (BD Horizon, Cat#562428) cells were analyzed for activation markers CD25 (Miltenyi Biotech, Cat#130-113-283) and CD69 (BD Pharmingen, Cat#555533) by flow cytometry. Supernatants were collected and stored at -80°C for cytokine release analysis by enzyme-linked immunosorbent assay (ELISA).

Tatari N., Zhang X., Chafe S.C., McKenna D., Lawson K.A., Subapanditha M., Shaikh M.V., Seyfrid M., Savage N., Venugopal C., Moffat J, & Singh S.K. (2022). Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors. Frontiers in Immunology, 13, 905768.

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Isolation and Activation of Human T Cells

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Ficoll density gradient (Ficoll-Paque PLUS; cat. #17144003) and RosetteSep Human T Cell Enrichment Cocktail (Stemcell; cat. #15021) were used according to the manufacturer’s protocol to isolate T cells by negative selection from the blood of healthy donors. Donors signed a clinical consent form for the donation procedure which includes language that the donor center can direct use of all materials and byproducts for clinical or research use. T cells were activated using ImmunoCult Human CD3/CD28 T Cell Activator tetramers following the manufacturer’s recommended protocol (Stemcell; cat. #10971) and cultured using ImmunoCult-XF T Cell Expansion Medium (Stemcell; cat. #10981) in the presence of 100U/ml of IL2 (Peprotech; cat. #200–02). At the indicated times, T cells were stained with the following antibodies: Alexa 647 conjugated 1G7 antibody (anti-KIR3DL3 antibody, generated as described in Methods) or Alexa 647 conjugated Mouse IgG2b, κ isotype control (Biolegend; cat. #400330) at 5µg/ml; BV785 anti-human CD3 (Biolegend; cat. #344842); PE/Cyanine7 conjugated anti-human CD8 (Biolegend; cat. #344712); PE/Cyanine7 conjugated anti-human CD4 (Biolgend; cat. #317414); PE/Cyanine7 conjugated Mouse IgG2b, κ isotype (Biolegend; cat. #400325); PE/Cyanine7 conjugated Mouse IgG1, κ isotype (Biolegend; cat. #400125); and BV785 conjugated Mouse IgG1, κ isotype (Biolegend; cat. #400169).

Bhatt R.S., Berjis A., Konge J.C., Mahoney K.M., Klee A.N., Freeman S.S., Chen C.H., Jegede O.A., Catalano P.J., Pignon J.C., Sticco-Ivins M., Zhu B., Hua P., Soden J., Zhu J., McDermott D.F., Arulanandam A.R., Signoretti S, & Freeman G.J. (2020). KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1. Cancer immunology research, 9(2), 156-169.

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