Anti cd3 bv510

Manufactured by BD

Sourced in United States

The Anti-CD3 BV510 is a fluorochrome-conjugated antibody that specifically binds to the CD3 antigen expressed on the surface of T cells. It is used in flow cytometry applications for the identification and analysis of T cell populations.

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Anti-CD3 (541 citations) CD3-BV510 (17 citations) BV510-conjugated anti-CD3 (3 citations) BV510-conjugated anti-human CD3 (3 citations) ), anti-CD3-BV510 (HIT3a), (3 citations) Anti-human CD3-BV510 (2 citations)

24 protocols using anti cd3 bv510

Multiparametric Murine Lymphocyte Profiling

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Cryopreserved mouse splenocytes were thawed and stained using the following Abs: FITC anti-CD4 (clone no. RM4-5, catalog no. 553047; 1:400 dilution; Becton Dickinson [BD]), PE anti-CD25 (clone no. 7D4, catalog no. 558642, 1:1000 dilution; BD), PE-CF594 anti-CD279 (PD-1) (clone no. J43, catalog no. 562523, 1:500 dilution; BD), biotin anti-CXCR5 (clone no. 2G8, catalog no. 551960, 1:100 dilution; BD), Alexa Fluor (AF) 700 anti-CD8 (clone no. 53-6.7, catalog no. 564983, 1:200 dilution; BD), Brilliant Violet (BV) 421 anti-CD127 (clone no. SB/199, catalog no. 566300, 1:100 dilution; BD), BV510 anti-CD3 (clone no. 145-2C11, catalog no. 563024, 1:100 dilution; BD), BV650 anti-NK1.1 (clone no. PK136, catalog no. 108740, 1:100 dilution; BioLegend), BV786 anti-B220 (clone no. RA3-6B2, catalog no. 563894, 1:200 dilution; BD), and streptavidin-AF647 (catalog no. 405237, 1:1500 dilution; BioLegend). B cells (CD3⁻B220⁺), NK cells (CD3⁻NKU⁺), CD8⁺ T cells (CD3⁺CD8⁺), Tfh cells (CD3⁺CD4⁺CD8⁻CD127^highCD25^lowPD-1⁺CXCR5⁺), and T follicular regulatory (Tfr) cells (CD3⁺CD4⁺CD8⁻CD127^lowCD25^highPD-1⁺CXCR5⁺) were sorted on a BD FACSAria Flow Cytometer.

Li D., Bradley T., Cain D.W., Pedroza-Pacheco I., Aggelakopoulou M., Parks R., Barr M., Xia S.M., Scearce R., Bowman C., Stevens G., Newman A., Hora B., Chen Y., Riebe K., Wang Y., Sempowski G., Saunders K.O., Borrow P, & Haynes B.F. (2020). RAB11FIP5-Deficient Mice Exhibit Cytokine-Related Transcriptomic Signatures. ImmunoHorizons, 4(11), 713-728.

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Flow Cytometry Analysis of Activated PBMCs

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For flow cytometry analysis, PBMCs (10⁶/tube) were stimulated with 50 ng/ml phorbol 12- myristate 13-acetate (PMA) and 1.0 mg/ml of ionomycin (Sigma, St. Louis, MO, USA) in 10% fetal bovine serum (FBS)-RPMI 1640 medium at 37 °C in a humidified incubator at 5% carbon dioxide for 2 h. Cells were cultured in the presence of 0.5 mg/mL brefeldin A (BFA, Sigma) for 4 h, harvested, stained with APC-H7-anti-CD4, BV510-anti-CD3, FITC-anti-CD154, FITC-anti-CD138, PE-Cy7-anti-CD19, PE-Cy7-anti-CD28, BV421-anti-PD1, PE-anti-ICOS and APC-anti-IL21, (BD Biosciences, San Diego, CA), washed with buffer (1 ml per tube; BD Biosciences, San Diego, CA), and centrifuged (250 × g), according to the manufacturer’s, instructions. Controls were isotype-matched antibodies. Cells were washed with PBS and subjected to flow cytometry analysis using a FACSAria II. At least 50,000 events per sample were analyzed using FlowJo software (v5.7.2)¹⁵.

Wang X., Li T., Si R., Chen J., Qu Z, & Jiang Y. (2020). Increased frequency of PD-1hiCXCR5- T cells and B cells in patients with newly diagnosed IgA nephropathy. Scientific Reports, 10, 492.

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Macrophage Activation Assay Protocol

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Lactate oxidase (LOX), N-(3-dimethylamino propyl-N′-ethylcarbodiimide) hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). CB839 was brought from APExBIO (Houston, TX, USA). Moreover, FITC-anti-CD86, PE-anti-CD206, APC-anti-CD40, APC-Cy7-anti-CD45, BV510-anti-CD3, FITC-anti-CD4, PE-anti-CD8, BV421-anti-F4/80, FITC-anti-CD11b, PE-anti-CD86, APC-anti-CD206, BV605-anti-Ly-6G, PE-Cy7-anti-Ly-6C, antibodies were supplied by BD Biosciences (San Jose, CA, USA). Other reagents were standard Sinopharm Chemical Reagent (Shanghai, China).

Feng Q., Hao Y., Yang S., Yuan X., Chen J., Mei Y., Liu L., Chang J., Zhang Z, & Wang L. (2022). A metabolic intervention strategy to break evolutionary adaptability of tumor for reinforced immunotherapy. Acta Pharmaceutica Sinica. B, 13(2), 775-786.

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Multiparametric Murine Lymphocyte Profiling

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Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells

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For flow cytometry analysis, harvested tumors were minced into small pieces with scissors and incubated in a digestion buffer at 37 °C for 70 min. The suspensions were filtrated using a cell strainer (BD Falcon, New Jersey, USA) and centrifuged at 2000 rpm for 5 min. The collected cells were re-suspended in ACK Lysis Buffer to remove red blood cells. Next, the obtained cells were resuspended in PBS and stained by Fixable Viability Dye eFluor^TM 780 (Thermo Fisher Scientific Co., Ltd., Shanghai, China) to exclude dead cells. Then, cells were washed with PBS and stained with anti-CD16/CD32 antibody (BD, New Jersey, USA) for Fc blocking to prevent nonspecific binding. Subsequently the cells were incubated with Percp-Cy5.5-anti-CD45, BV510-anti-CD3, FITC-anti-CD4, BV421-anti-CD8, PE-anti-B220, APC-anti-CD11c, FITC-anti-CD11b, PE-anti-F4/80, BV650-anti-CD86 and PE-Cy7-anti-CD206 (BD, Shanghai, China) to specifically label the T lymphocytes, B lymphocytes, DCs and Macrophage. Expect Fixable Viability Dye eFluor^TM 780 and anti-CD16/32. All the above staining and centrifugation processes were carried out at 4 °C under darkness. The samples were analyzed by FCM (Sony ID7000™, Tokyo Metropolis, Japan).

Xu H., Qin X., Guo Y., Zhao S., Feng X., Zhang R., Tian T., Kong L., Yang C, & Zhang Z. (2024). Radiation-based immunogenic vaccine combined with a macrophage “checkpoint inhibitor” for boosting innate and adaptive immunity against metastatic colon cancers. Acta Pharmaceutica Sinica. B, 14(5), 2247-2262.

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Multiparametric Immune Cell Analysis

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Splenocytes and lung cells were incubated with anti-CD16/32 (BD Bioscience, USA) for 30min at 4°C in PBS supplemented with 2% FBS (FACS buffer) to block non-specific binding with Fc receptors. Cells were surface-stained with BV650-anti-CD8 (BD-563234, USA), BV711-anti-CD4 (BD-563050, USA), BV510anti-CD3 (BD-563024, USA), PECY7-anti-CD25 (eBioscience-25-0251-82, USA), PE-anti-F4/80 (BD-565410, USA), BV510-anti-CD11b (BD-562950, USA), APC-anti-CD11c (eBioscience-17-0114-81, USA) for 20 min on ice and washed twice with PBS. For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD Bioscience, USA) and stained with APC-anti-IL-4 (eBioscience-17-7041-82, USA), PE-anti-IFN-γ (eBioscience-12-7311-82, USA), PE-anti-IL-2 (eBioscience-12-7021-82, USA), BV421-anti-IL-17 (BD-563354, USA), PECY7-anti-TNF-ɑ (BD-557644, USA), Pacific blue-anti-Foxp3 (eBioscience-48-5773-82, USA) for 20min. Cells were washed with 1×BD Perm/Wash buffer and were finally resuspended in 4% paraformaldehyde (PFA) for flow cytometry analysis. Flow cytometry was performed using BD LSR Fortessa (BD Bioscience, USA) and raw data was analyzed using FlowJo software.

Jia R., Liu S., Xu J, & Liang X. (2020). IL16 deficiency enhances Th1 and cytotoxic T lymphocyte response against influenza A virus infection. Bioscience trends, 13(6).

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Multicolor Flow Cytometry Analysis of Tfh Cells

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Freshly isolated PBMCs (4 × 10⁶/mL) were cultured in 10 % fetal calf serum RPMI-1640 (Hyclone, Logan, UT, USA) in U-bottom 24-well tissue culture plates (Costar, Lowell, MA, USA), stimulated with or without 50 ng/mL of phorbol myristate acetate (PMA) plus 2 μg/mL of ionomycin (Sigma, St. Louis, MO, USA) for one hour, followed by treatment with Brefeldin A (10 μg/mL, GolgiStop™; BD Biosciences, San Jose, CA, USA) for an additional five hours. Then, these cells were stained in duplicate with BV510-anti-CD3, APC-H7-anti-CD4, BB515-anti-CXCR5, PE-Cy5-anti-CD45RA, PE-CF594-anti-CD279 and BV421-anti-CD278 (Beckton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Subsequently, cells were fixed, permeabilized, and stained with PE-anti-IL-21 (Beckton Dickinson). The frequencies of distinct Tfh cells were analyzed by multicolor flow cytometry (FACSAria™ II, BD Biosciences), and data were processed using FlowJo software (v5.7.2; FlowJo, Ashland, OR, USA).

Liu D., Liu J., Wang J., Liu C., Yang S, & Jiang Y. (2016). Distinct phenotypic subpopulations of circulating CD4+CXCR5+ follicular helper T cells in children with active IgA vasculitis. BMC Immunology, 17, 40.

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Peripheral Blood Mononuclear Cell Isolation and Analysis

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Blood samples were collected separately from controls and KD patients in different phases. Peripheral blood mononuclear cells (PBMCs) were isolated from individual KD patients and control subjects by density-gradient centrifugation at 800×g for 30 min at 25 °C using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). Freshly isolated PBMCs (4 × 10⁶/mL) were cultured in 10% fetal calf serum RPMI-1640 (Hyclone, Logan, UT, USA) in U-bottom 24-well tissue culture plates (Costar, Lowell, MA, USA) and stimulated for 1 h with or without 50 ng/mL of phorbol myristate acetate (PMA) in the presence of 2 μg/mL of ionomycin (Sigma, St. Louis, MO, USA). The cells were then treated with Brefeldin A (10 μg/mL, GolgiStop™, BD Biosciences, San Jose, CA, USA) for an additional 5 h. For flow cytometric analysis, PBMCs were stained in duplicate with BV510-anti-CD3, APC-H7-anti-CD4, BB515-anti-CXCR5, PE-Cy5-anti-CD45RA, PE-CF594-anti-CD279 and BV421-anti-CD278 (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Subsequently, the cells were fixed, permeabilized, and stained with PE-anti-IL-21 (Becton Dickinson). Multicolor flow cytometry (FACSAria™ II, BD Biosciences) was used to determine the percentages of distinct cTfh cells, and the data were analyzed with FlowJo software (v5.7.2; FlowJo, Ashland, OR, USA).

Xu M., Jiang Y., Zhang J., Zheng Y., Liu D., Guo L, & Yang S. (2018). Variation in IL-21-secreting circulating follicular helper T cells in Kawasaki disease. BMC Immunology, 19, 43.

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Multicolor Flow Cytometry Analysis of PBMCs

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PBMCs at 4 × 10⁶/ml were analyzed by multicolor flow cytometry (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA). Human PBMCs (10⁶/tube) were stained with BV510-anti-CD3 (clone: UCHT1), allophycocyanin (APC)-H7-anti-CD4 (clone: RPA-T4), BB515-anti-CXCR5 (clone: RF8B2), phycoerythrin (PE)-Cy5-anti-CD45RA (clone: HI100), PE-Cy7-anti-CCR6 (clone: 11A9), or APC-anti-CD183 (clone: IC6/CXCR3) (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Data were processed using FlowJo v.5.7.2 software (Tree Star, Ashland, OR, USA).

Liu D., Liu J., Wang J., Guo L., Liu C., Jiang Y., Wang H, & Yang S. (2017). Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children. PLoS ONE, 12(12), e0189133.

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Multiparameter Analysis of Colon Immune Cells

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Single cell suspension of colon cells were isolated as previously described [41 (link), 42 (link)] and analyzed by flow cytometry. Cells were stained with the following antibody panels: panel 1) CD45, CD11b, CD11c, F4/80, MHC II, Ly-6C, and Ly-6G; panel 2) CD45, CD11b, CD3, CD4, CD8, CD19, and MHC II. Flow cytometry was performed with the FACS LSR Fortessa (BD Biosciences) and data were analyzed using DIVA software (BD Biosciences). The following antibodies were used: anti-F4/80 BV421 (cat. 565,411), anti-Ly-6C Alexa Fluor 700 (cat. 561,237), anti-CD11c PE (cat. 561,044), anti-MHC II BV650 (cat. 563,415), anti-CD3 BV510 (cat. 563,024) from BD Bioscences (Milano, Italy); anti-CD11b PerCP-Cy5.5 (cat. 45–0112), anti-CD4 Alexa Fluor 700 (cat. 56–0041), anti-Ly-6G FITC (cat. 11–5931), anti-CD45 Pe-Cy5 (cat. 15–0451), anti-CD8 PE (cat. 12–0083), anti-CD19 FITC (cat. 11–0193) from EBiosciences (San Diego, CA, USA).

Andreuzzi E., Fejza A., Polano M., Poletto E., Camicia L., Carobolante G., Tarticchio G., Todaro F., Di Carlo E., Scarpa M., Scarpa M., Paulitti A., Capuano A., Canzonieri V., Maiero S., Fornasarig M., Cannizzaro R., Doliana R., Colombatti A., Spessotto P, & Mongiat M. (2022). Colorectal cancer development is affected by the ECM molecule EMILIN-2 hinging on macrophage polarization via the TLR-4/MyD88 pathway. Journal of Experimental & Clinical Cancer Research : CR, 41, 60.

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