Cd3 buv395

Manufactured by BD

Sourced in United States

The CD3-BUV395 is a fluorescent antibody that recognizes the CD3 protein, a component of the T cell receptor complex. It is used in flow cytometry applications to identify and analyze T cells.

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Lab products found in correlation

Cd4 buv496, by BD (6 mentions) Pd 1 bv711, by BioLegend (4 mentions) Cd25 pe cy7, by BioLegend (3 mentions) Percp cy5.5 cd45ro, by BioLegend (3 mentions) Cd45ra pe, by BioLegend (3 mentions) Cd4 bv421, by BioLegend (3 mentions) Cd4 bv510, by BD (3 mentions) Cd14 apc cy7, by BD (3 mentions) Cd38 pe cy7, by BioLegend (3 mentions) Cd19 bv510, by BioLegend (3 mentions) Cd19 apc cy7, by BD (3 mentions) Cd3 apc cy7, by BioLegend (3 mentions) Cd10 pe dazzle594, by BioLegend (3 mentions) Cd21 apc, by BioLegend (3 mentions) Igd buv 737, by BD (3 mentions) Igg percp, by BD (3 mentions) Cxcr5 pe cy7, by BD (3 mentions) Iga pe, by Miltenyi Biotec (3 mentions) Cd27 bv650, by BioLegend (3 mentions) Cd25 bv421, by BD (3 mentions)

24 protocols using cd3 buv395

Flow Cytometry Immunophenotyping of PBMCs

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PBMCs were stained for 30 min at 4°C with antibodies to BUV395–CD3, BV510–CD8α, PE–cyanin 5 (Cy5)–CD45RA, PE–Cy7–CCR7 (all from BD Biosciences), and Pacific Blue–IL–7Rα (Biolegend,). Stained cells were washed with PBS, fixed for 20 min with 2% paraformaldehyde (Biolegend), and acquired (> ~3 × 10⁵ cells) using an LSRII flow cytometer (BD Biosciences). Flow cytometric data were analyzed using FlowJo software (FlowJo, LLC,).

Shin M.S., Park H., Salahuddin S., Montgomery R.R., Emu B., Shaw A.C, & Kang I. (2022). Alterations in high‐dimensional T‐cell profile and gene signature of immune aging in HIV‐infected older adults without viremia. Aging Cell, 21(10), e13702.

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Multiparametric Immunophenotyping of Human PBMCs

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Human PBMCs were stained with BV421-CD4 (BioLegend, OKT4, CAT# 304222, RRID: AB_2562134), PE/Cy7-CD25 (BioLegend, M-A251, CAT# 356108, RRID: AB_2561975), PE-CD45RA (BioLegend, HI100, CAT# 304108, RRID: AB_314412), PerCP/Cy5.5-CD45RO (BioLegend, UCHL1, CAT# 304222, RRID: AB_2174124), BUV395-CD3 (BD Biosciences, UCHT1, CAT# 563548, RRID: AB_2744387), APC-FOXP3 (Thermo Scientific, PCH101, CAT# 17-4776-42, RRID: AB_1603280), FITC-Ki67 (BD, B56, CAT# 556026, RRID: AB_396302). T cells activation with phorbol-12-myristate 13-acetate/ionomycin (PMA/iono) was performed according to the manufacturer’s protocol (BioLegend, CAT# 423301).

Nagle V.L., Hertz C.A., Henry K.E., Graham M.S., Campos C., Pillarsetty N., Schietinger A., Mellinghoff I.K, & Lewis J.S. (2022). Non-invasive imaging of CD4+ T cells in humanized mice. Molecular cancer therapeutics, 21(4), 658-666.

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Phenotyping Brain-Resident Immune Cells

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Following dissection, whole brains were mechanically homogenized and passed through a 100-μM sterile filter. Cells were treated with a CNS-Specific Percoll Isotonic solution (Percoll – Cytiva) to remove myelin. Brain cells were stained with BV421-CD4 (BioLegend, OKT4, CAT# 304222, RRID: AB_2562134), PE/Cy7-CD25 (BioLegend, M-A251, CAT# 356108, RRID: AB_2561975), PE-CD45RA (BioLegend, HI100, CAT# 304108, RRID: AB_314412), PerCP/Cy5.5-CD45RO (BioLegend, UCHL1, CAT# 304222, RRID: AB_2174124), BUV395-CD3 (BD Biosciences, UCHT1, CAT# 563548, RRID: AB_2744387), APC-FOXP3 (Thermo Scientific, PCH101, CAT# 17-4776-42, RRID: AB_1603280) and LIVE/DEAD Fixable Green Dead Cell Stain Kit (ThermoFisher Scientific, CAT# L23101P).

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Isolation and Characterization of CD4+ T Cells

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CD4+ cells were isolated from bulk PBMCs using the CD4+ T Cell Isolation Kit with MicroBeads for column-based magnetic activated cell sorting (MACS)(Miltenyi Biotech, CAT# 130-096-533). MACS was performed according to the manufacturers protocol. Cells were stained with PE/Cy7-CD25 (BioLegend, M-A251, CAT# 356108, RRID: AB_2561975), PE-CD45RA (BioLegend, HI100, CAT# 304108, RRID: AB_314412), PerCP/Cy5.5-CD45RO (BioLegend, UCHL1, CAT# 304222, RRID: AB_2174124), BUV395-CD3 (BD Biosciences, UCHT1, CAT# 563548, RRID: AB_2744387), and LIVE/DEAD Green Dead Cell Stain Kit (ThermoFisher Scientific, CAT# L23101). Pooled cells from all groups were used to generate the LIVE/DEAD single-color control, all fluorescence minus one (FMO) controls, and unstained controls. Ultracomp eBeads (ThermoFisher Scientific, CAT# 01-2222-41) were stained to generate single color controls. Analysis was performed using FlowJo (Version 10, RRID: SCR_008520). FACs gating strategy is detailed in Supplemental Figure 1.

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Influenza Vaccine-Induced T Cell Responses

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Between October and February during the 2020–2021 influenza season, peripheral blood samples were collected before vaccination and at a mean of 32 days (range: 27–64 days) after vaccination. Peripheral blood mononuclear cells (PBMCs) were prepared from blood on FicollPAQUE gradients. To identify the distribution of T cell subsets, the cells were stained with antibodies to BUV395-CD3, Alexa Fluor 700- or isotype antibodies. For intracellular cytokine staining, PBMCs were incubated for 6 hours with or without a combination of Quadrivalent Influenza Vaccine (Seqirus, Parkville, Australia) and anti-CD28/49d costimulatory antibodies (BD Biosciences) in the presence of Golgiplug (BD Biosciences). Incubated cells were stained with antibodies to BUV395-CD3 and Alexa Fluor 700-CD4 (all from BD Biosciences). Cells were fixed and permeabilized using Foxp3/transcription factor staining buffer set according to the manufacturer’s protocol (Invitrogen, Waltham, MA) and stained with antibodies to APC-Cy7- IFN-γ and APC-TNF-α (Biolegend, San Diego, CA). Cells were analyzed using an LSRII® flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).

Leone D.M., Park H.J., Unlu S., Gurvitz M., Kang I, & Elder R.W. (2022). T Cell Response to Influenza Vaccination Remains Intact in Adults with Congenital Heart Disease Who Underwent Early Thymectomy. International journal of cardiology. Congenital heart disease, 8, 100359.

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Characterization of Murine Lymphoid Cells

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Draining s.c. LNs (axillary and popliteal) and the spleen were isolated from mice. Spleens were first mashed into a single cell suspension with plain DMEM media (Gibco 11966025), filtered through 70 μM cell strainers, and lysed with ACK lysis buffer (Gibco A1049201). LNs were digested with 1 mg/mL Ca²⁺ supplemented Collagenase D (Roche 11088866001) for 45 min at 37°C and gently mashed into a single cell suspension, also with DMEM and through 70 μM cell strainers. The cells were resuspended in IMDM media (Gibco 12440053), supplemented with 10% FBS and 1% Penicillin-Streptomycin (Gibco 15140122), and counted using a LUNA automated fluorescent cell counter (Logos biosystems). Cells were seeded at a count of 1x10⁶ - 3x10⁶ per well in 96-well round-bottom plates for subsequent antibody staining for flow cytometry. Antibodies against the following markers were used: CD3 – BUV395 (BD Biosciences 563565), CD8-BUV737 (BD Biosciences 612759), CD4 – BUV496 (BD Biosciences 612952), Foxp3 – FITC (BD Biosciences 560403), CD25 – BV605 (BioLegend 120235), ST2 – BV421 (BD Biosciences 566309), Lag3 – PerCP-Cy5.5 (BD Biosciences 564673), CTLA4 – PE-Cy7 (eBioScience 17-1522-82), IFNγ – APC (BioLegend 505810), TNFα – BV605 (BioLegend 506329), IL-2 – FITC (BioLegend 503806), IL-10 – APC-Cy7 (BioLegend 505036), PD-1 – BV711 (BioLegend 135231), Tim3 – PE (BD Biosciences 566346).

Maulloo C.D., Cao S., Watkins E.A., Raczy M.M., Solanki A.S., Nguyen M., Reda J.W., Shim H.N., Wilson D.S., Swartz M.A, & Hubbell J.A. (2021). Lymph Node-Targeted Synthetically Glycosylated Antigen Leads to Antigen-Specific Immunological Tolerance. Frontiers in Immunology, 12, 714842.

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Multicolor Flow Cytometry Panel for T-Cell and Macrophage Immunophenotyping

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For staining T-cells and M1 & M2 cells, 100 µl (approx. one million cells) of PBMCs suspended in PBS were stained for T cells by adding titrated volume of BD Horizon™ surface antibodies i.e., CD3-BUV395 (Cat. 564001), CD4-BV510 (Cat. 562970), CD8-BV605 (Cat. 564116), CD45RA-BB515 (Cat. 564552), CD45RO-APC (Cat. 559865), CD197-BB700 (Cat. 566437), CD16-PE (Cat. 555407), CD56-PE (Cat. 555516), CD19-AF700 (Cat. 566946), and for M1 and M2 cells, CD68-APC (Cat. 562111), CD80-PE (Cat. 557227), CD86-BV510 (Cat. 563461), CD163-BV421 (Cat. 562643), CD206-BB515 (Cat. 564668), to each sample tube and incubated in dark for 15–30 min at room temperature. Following incubation, cells were washed thrice with 2 ml sheath fluid by spinning at 200–300 g for 3–5 min. Finally washed pellet was re-suspended in 500 µl of sheath. 50,000 cells were acquired using BD LSR Fortessa flow cytometer.

Kumari D., Singh S., Kumari M., Gupta H., Chauhan D., Singh K., Rao Eslavath M., Bhushan B., Dogra V., Bargotya M., Bhattar S., Gupta U., Hussain J., Jain S., Meena R., Varshney R., Ganju L, & Singh Y. (2023). Flow cytometry profiling of cellular immune response in COVID-19 infected, recovered and vaccinated individuals. Immunobiology, 228(3), 152392.

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Comprehensive B and T Cell Profiling

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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Krishnananthasivam S., Li H., Bouzeyen R., Shunmuganathan B., Purushotorman K., Liao X., Du F., Friis C.G., Crawshay-Williams F., Boon L.H., Xinlei Q., Chan C.E., Sobota R., Kozma M., Barcelli V., Wang G., Huang H., Floto A., Bifani P., Javid B, & MacAry P.A. (2023). An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis. NPJ Vaccines, 8, 127.

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Comprehensive T-cell Immunophenotyping Protocol

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Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).

Wu T., Tan J.H., Sin W., Luah Y.H., Tan S.Y., Goh M., Birnbaum M.E., Chen Q, & Cheow L.F. (2023). Cell Granularity Reflects Immune Cell Function and Enables Selection of Lymphocytes with Superior Attributes for Immunotherapy. Advanced Science, 10(28), 2302175.

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Comprehensive Immune Cell Analysis

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The following antibodies were used for flow cytometry analysis: CD45-FITC (Cat. # 553079; BD Biosciences), CD3-BUV395 (Cat. # 563565; BD Biosciences), CD4-BUV737 (Cat. # 612761; BD Biosciences), CD8-Percp-Cy5.5 (Cat. # 45-0081-82; eBioscience), CD44-BV711 (Cat. # 103057; Biolegend), CD335-PE/Dazzle594 (Cat. #137630; Biolegend), PD-1-PE (Cat. # 551892; BD Biosciences), Ki67*-BV605 (Cat. # 652413; Biolegend), Granzyme B*-APC (Cat. # 366408; Biolegend), IFN-γ*-BV421 (Cat. # 563376; BD Biosciences), CD11b-PE-Cy7 (Cat. # 101216; Biolegend), F4/80-BV510 (Cat. # 123135; Biolegend), CD206-AF700 (Cat. # 141734; Biolegend), I-A/I-E-BV786 (Cat. # 743875; BD Biosciences), and L/D-efluor780 (Cat. # 65-0865-18; eBioscience).

Zhang Y., Gabere M., Taylor M.A., Simoes C.C., Dumbauld C., Barro O., Tesfay M.Z., Graham A.L., Ferdous K.U., Savenka A.V., Chamcheu J.C., Washam C.L., Alkam D., Gies A., Byrum S.D., Conti M., Post S.R., Kelly T., Borad M.J., Cannon M.J., Basnakian A, & Nagalo B.M. (2022). Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy. Frontiers in Oncology, 12, 1042250.

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