Splenocytes were stained according to the manufacturer’s guidelines with CD4-BUV395, CD8-BV786, CD44-APC-Cy7, KLRG-1-V450, IL-2-PE (BD Pharmingen), LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific), and CD-127-PE-Cy7, CD3-BV711, Thy1.1-A700, IFN-γ-PE/Dazzle594 (BioLegend). Flow cytometry was performed using a BD LSR Fortessa Cell Analyzer (BD Biosciences, San Jose, CA). Data collected were analyzed using FlowJo Software (Tree Star, San Carlos, CA) and analyzed with Prism 6 software (GraphPad Software Inc).
Cd3 bv711
Manufactured by BioLegend
Sourced in United States
CD3-BV711 is a fluorochrome-conjugated antibody used for the detection and analysis of CD3-positive cells in flow cytometry applications. It binds to the CD3 complex, which is expressed on T cells, and the BV711 fluorochrome provides a specific signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa cell analyzer, by BD (2 mentions)
Cd8 bv786, by BD (2 mentions)
Cd8 apc cy7, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Zombie aqua, by BioLegend (2 mentions)
Cd56 bv510, by BioLegend (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Cd45 fitc, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Cd127 pe cy7, by BioLegend (1 mentions)
Il 2 pe, by BD (1 mentions)
Cd44 apc cy7, by BD (1 mentions)
Ifn γ pe dazzle594, by BioLegend (1 mentions)
Cd4 buv395, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Live dead, by Thermo Fisher Scientific (1 mentions)
Cd14 bv711, by BioLegend (1 mentions)
Cd38 v450, by BD (1 mentions)
Cd138 apc, by Miltenyi Biotec (1 mentions)
11 protocols using cd3 bv711
1
Multicolor Flow Cytometry Staining
2
Isolation and Purification of Antibody-Secreting Cells
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PBMCs and BM mononuclear cells were isolated from fresh blood and BM aspirate samples, respectively, by density gradient centrifugation using Lymphocyte Separation Medium (Cellgro/Corning). After isolation, the mononuclear cell fractions were further enriched by negative selection using a MACS column that removed CD3+/CD14+ cells, a commercial human pan-B cell enrichment kit that removes cells expressing CD2, CD3, CD14, CD16, CD36, CD42b, CD56, CD66b, CD123, and glycophorin A, or a custom stem cell kit that removes CD66b+/GPA+/CD3+/CD14+ cells (StemCell Technologies). Enriched fractions were then stained using the fluorescently conjugated antibodies IgD–FITC (Cat. no. BD555778; BD Biosciences), CD3-BV711 (Cat. no. 317328; BioLegend), CD14-BV711 (Cat. no. 301838; BioLegend), CD19-PE-Cy7 (Cat. no. 301838; BD Biosciences), CD38-V450 (Cat. no. BDB561378; BD Biosciences), CD138-APC (Cat. no. 130-117-395; Miltenyi Biotech), CD27-APC-e780 (Cat. no. 5016160; eBiosciences), and LiveDead (L34966; Invitrogen) and populations of ASC were purified by fluorescent activated cell sorting for further experiments. Cell sorting experiments were performed on a FACS Aria II (BD Biosciences) using a standardized sorting procedure that used rainbow calibration particles to ensure consistency of sorts over time. The gating strategies used to isolate ASC populations are in Figs S1 and S6 .
3
Comprehensive Immune Cell Profiling
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The adipose SVCs and splenocytes were incubated for 10 minutes with CD16/32 antibody (FC block) (eBioscience) and then stained with other fluorochrome-conjugated antibodies for 20 minutes at 4℃. All cells were stained with antibodies against CD45-PE/Cy7 (eBioscience), CD4-BV500 (BD Biosciences), CD8-APC/Cy7 (Biolegend), CD19-PE (Biolegend), NK1.1-BV421 (Biolegend), NKp46-PE/Dazzle594 (Biolegend), CD11b-BV786 (Biolegend), CD11c-APC (Biolegend), CD3-BV711 (Biolegend), and F4/80-BV650 (Biolegend). 7AAD (Invitrogen) was used for live/dead staining. The stained cells were washed and analyzed with an LSRFortessa flow cytometer (BD Biosciences). NK cells were defined as CD45+ F4/80− CD19− CD3− NK1.1+ in lymphocyte gating. The NKp46+ and GFP+ cells were then gated. ATMs were defined as CD45+ CD19− CD3− NK1.1− F4/80+ CD11b+. Total cell numbers were counted with a hemocytometer and normalized according to the tissue weight (cells/g). Flow cytometric data were analyzed by using the FlowJo software (Flow Jo).
4
Characterizing Immune Cell Activation
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Fresh PBMCs were cultured either alone, with Pam3CysSK4 or XS15, or a mix of phytohaemagglutinin-L (PHA) and Pokeweed mitogen (PWM). Adherent and non-adherent cells were stained with mAbs: CD3-BV711, CD56-BV421, CD19-BV785, Zombie aqua (all Biolegend), CD14-Alexa Fluor 700, HLA-DR-PerCP, and CD69-APC-Cy7 (all BD). Cells were measured by flow cytometry as described above.
5
Profiling Natural Killer Cells in Sarcoidosis
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PBMCs were isolated from SLT patients (n = 16) and HCs (n = 17), and fresh cells were stained for CD45 (CD45-FITC), CD3 (CD3-BV711), CD56 (CD56–BV510), and CD16 (CD16-PE/Cy7; BioLegend, cat#302016), and analysed via FACS. CD45+CD3−CD56+CD16+ (NK) cells as a proportion of CD45+ cells were calculated, as were the ratio of CD56+CD16+ to CD56+CD16− cells. PBMCs were also stimulated with IL-12 (50 ng/ml; Peprotech, cat#200-12) and IL-18 (50 ng/ml; Cambridge bioscience, cat#230-00229-10) in the presence of brefeldin A (5 μg/ml; Sigma, cat#B7651) for 4 h and stained for viability, CD45, CD3, CD56, and IFNγ. The proportion of viable CD45+CD3−CD56+ cells expressing IFNγ was determined. Stimulation was also performed in HCs (n = 8) in media supplemented with 40 mM NaCl. The proportion of NK cells (CD45+CD3−CD56+) was determined as was the expression of IFNγ by CD56+ cells, and compared between standard and high salt conditions.
6
Quantitative Analysis of TNFR2 Expression
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The immunochemical reagents used were as follows: anti-human IgG, Fcγ-PE (1/200 dilution, #109-116-170; Jackson Immunoresearch, West Grove, PA, USA), CD3-BV711 (1/250 dilution, #300463; BioLegend, San Diego, CA, USA), CD4-BV510 (1/100 dilution, #344633; BioLegend), CD25-BV421 or CD25-BV605 (1/40 dilution, #302629 or #302631; BioLegend); CD127-PerCP-Cy5.5 (1/40 dilution, #560551; BD Biosciences, Franklin Lakes, NJ, USA), TNFR2-PE (1/70 or 1/100 dilution, #22235; R&D Systems, Minneapolis, MN, USA), and Foxp3-AlexaFluor 647 (1/100 dilution, #320213; BioLegend). Cells were treated with PBS containing 0.2% sodium azide and 5% FBS. For the antibody-binding analysis of TNFR2-expressing Ramos-Blue and HEK293T cells, primary antibodies were first incubated in a dilution series for 30 min on ice and then labeled with anti-human IgG-PE. For quantitation, BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences, lot #76536) was used. For the multi-color labeling experiment, fluorescent intensities were compensated using BD™ CompBeads Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences), and the performance of two anti-CD25 antibodies was confirmed to be equivalent. The cells were analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) and data was analyzed using FlowJo_v10.8.1 (FlowJo, LLC).
7
Multiparameter Flow Cytometry for Immune Profiling
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The following monoclonal antibodies and reagents were used with the appropriate isotype controls and were obtained from BD Biosciences unless otherwise stated. Human immunoglobulin κ light chain allophycocyanin (APC) (catalog no. 561323), CD45 AlexaFluor 700 (catalog no. 56056), CD19 APC-Cy7 (Biolegend, catalog no. 348794), human immunoglobulin λ light chain AlexaFluor 488 (Biolegend, catalog no. 316612), CD138 PerCP-Cy5.5 (catalog no. 564605), CD38 BV421 (catalog no. 562444), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, catalog no. L34957), CD14 V500 (catalog no. 561391), CD56 BV605 (catalog no. 562780), CD20 BV650 (catalog no. 563780), CD3 BV711 (Biolegend, catalog no. 317328), BCMA PE (Biolegend, catalog no. 357504), CD200 PE-Cy7 (Biolegend, catalog no. 329212), CD200 PE (Biolegend, catalog no. 329306), CD200R PE-Cy7 (Biolegend, catalog no. 329312), CD200R APC (Biolegend, catalog no. 329307), CD200R PE (Biolegend), CD3 BV421 (catalog no. 562427), CD4 BV785 (Biolegend, catalog no. 317442), CD8 APC-Cy7 (catalog no. 557834), CD197(CCR7) FITC (catalog no. 561271), CD45RO PE-Cy7(Biolegend, catalog no. 304230), and CD274(PD-L1) APC (catalog no. 563741). The data were acquired with BD Fortessa and analyzed with FlowJo (version 10).
8
Murine Lung Immune Cell Profiling
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On day 7, the lungs were isolated from mice, grounded, and subsequently passed through a 70μm cell strainer. Ammoniumchloride-potassium lysing buffer was used for erythrocyte lysis. Multiparameter assessments were performed using BD FACSAria II (BD Biosciences) and data was analyzed with Flowjo software (version 10.6). After staining Zombie Aqua™ (Biolegend) to exclude dead cells, single-cell suspensions were incubated with the fluorochrome-conjugated antibodies in PBS containing 2% fetal bovine serum and then washed twice before detection. Antibodies specific to mouse used included CD16/32 (BD, 553,141), CD45-APC-Cy7 (Biolegend, 103,116), CD11b-AF700 (Biolegend, 101,222), Ly6G-PE (Biolegend, 127,608), CD62L-BV650 (Biolegend, 104,453), CD3-BV711 (Biolegend, 100,241), CD19-BV711 (Biolegend, 115,555), NK1.1-Pacific Blue (Biolegend, 109,816).
9
Identification of HIV-specific T follicular Helper Cells
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HIV-specific Tfh responses were defined using fluorochrome conjugated HLA class II tetramers. Briefly, cells were stained for 1 h at 37 °C with APC and PE conjugated HLA Class II tetramer complexes, washed in 2% FCS-PBS and then stained with these antibodies: LIVE/DEAD Fixable Blue dead cell stain kit (Thermo Fisher Scientific), CD3 BV711 (Biolegend), CD4 BV650 (BD Biosciences), CD8 BV786 (BD Biosciences), CXCR5 AF488 (BD Biosciences), CXCR3 BV605 (Biolegend), PD-1 BV421 (Biolegend) and CD45RA AF700 (Biolegend); for 20 min at RT. Cells were washed and acquired on the LSRFortessa (BD Biosciences).
10
Multiparametric Analysis of T-cell Responses
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ICS was performed as reported earlier [23 (link)]. Cells were stimulated with individual peptides or with an equal volume of water/ 10% DMSO in the presence of anti-CD107a (BD), GolgiStop (BD) and Brefeldin A (Sigma-Aldrich). After 12 h cells were stained for CD4-APC-Cy7 (BD), CD8-PECy7 (Beckman Coulter) and CD3-BV711 (Biolegend) and with Aqua Live Dead, fixed and permeabilized in (Cytoperm/Cytofix; BD) and further stained for IFNγ-Alexa Fluor 700 (BD Biosciences), anti-TNF-Pacific Blue (Biolegend), IL-10-PE and IL-2-APC (both BD).
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