Cell cytometry data is available in FlowRespository database (
Bv510
Manufactured by BD
Sourced in United States
The BV510 is a fluorescence-based flow cytometry reagent. It is designed to detect and analyze specific cellular markers or proteins expressed on the surface of cells.
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Lab products found in correlation
Bv421, by BD (9 mentions)
Bv605, by BD (4 mentions)
Pe cy7, by BD (4 mentions)
Apc cy7, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Apc h7, by BD (2 mentions)
Percp cy, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Bv650, by BD (2 mentions)
Facsaria instrument, by BD (1 mentions)
Fortessa analyser, by BD (1 mentions)
Pe cy7, by Thermo Fisher Scientific (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
70 μm nylon cell strainer, by BD (1 mentions)
Tnf α, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Alexa fluor 350 carboxylic acid succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Fitc anti cd117, by BioLegend (1 mentions)
20 protocols using bv510
1
Detailed Workflow for Cell Sorting and Flow Cytometry
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For cell sorting: 30 × 10E6 cells were counted and resuspended in 300 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 15 μl of the human anti-CD19 antibody conjugated with BV510 (Becton Dickinson, 562947) and 15 μl of human anti-cd11b (Mac1) antibody conjugated with PE-Cy7 (eBioscience, 25-0118-41) were added. Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 2 ml of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were sorted in a BD FACS Aria instrument at the Flow Cytometry Unit of the Centre for Genomic Regulation.
For flow cytometry analysis : 1 × 10E6 cells were counted and resuspended in 100 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 5 μl of each of the corresponding antibodies were added. For the CD19 knockout experiment, we used the antibody anti-CD19 conjugated with APC-Cy7 (Becton Dickinson, 557791). Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 500 ul of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were measured in a BD Fortessa analyser. For the Stain Index calculation we used the formula: (mean positive—mean background) / (2 * SD background), as previously described [63 (link)].
Cell cytometry data is available in FlowRespository database (https://flowrepository.org ) [64 (link)].
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Cell cytometry data is available in FlowRespository database (
2
Flow Cytometric Analysis of Dendritic Cell Markers
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Cells (collected from spleen and lymph node from 1 - and vehicle-treated mice, see above) were resuspended in 100 μl of FACS buffer (PBS, pH 7.2, containing 0.5% bovine serum albumin) and stained with appropriate conjugated antibodies for 30 min at 4 °C. To assess the migratory markers expression on DC, lymph nodes were collected at 7 dpi and disease onset (11 dpi) and passed through a 70-μm nylon cell strainer (Falcon) to prepare a single-cell suspension and used to optimize staining conditions. Lymph node cells were counted automatically (Countess, Invitrogen), and BV510-positive (Becton–Dickinson) DC (live cells) were gated APC Vio 770-conjugated anti-CD45, Pe Vio-770-conjugated anti-MHCII, and APC-conjugated anti-CD11c antibodies (diluted 1:100, Miltenyi) (see Additional file 1 ) and antibodies against relevant surface markers, PE-conjugated anti-CCR7, FITC-conjugated anti-CXCR4 (diluted 1:100, Miltenyi), PB-conjugated anti-CD40, or PerCP-conjugated anti-CD86 (diluted 1:100, Biolegend). Data were acquired on a FACS Canto II (Becton–Dickinson) and analyzed using DIVA 6.1 software.
3
Multiparametric Flow Cytometry Analysis of PBMCs
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PBMCs were then surface-stained with the following monoclonal antibodies to: CD3 (PerCP, Bio Legend, San Diego, USA), CD4 (Alexa Flour 700, Becton Dickinson, San Diego, CA, USA), CD8 (APC-H7, Becton Dickinson, San Diego, CA, USA), CD56 (FITC Becton Dickinson, San Diego, CA, USA). Next, monoclonal antibodies were washed out and stained for live/dead discrimination with an aminereactive dye (Alexa Fluor 350 carboxylic acid, succinimidyl ester, Invitrogen, Carlsbad, CA, USA). After 10 min of incubation at room temperature, cells were fixed and permeabilized using a fix and perm kit® (An Der Grub Bio Research GmbH, Vienna, Austria). Afterwards, intracellular staining was conducted with antibodies against interferon-γ (IFN-γ, BV605 Becton Dickinson, San Diego, CA, USA), interleukin-2 (IL-2, BV510 Becton Dickinson, San Diego, CA, USA) and tumor necrosis factor-α (TNF-α, BV421 Becton Dickinson, San Diego, CA, USA). Measurements were carried out on a flow cytometer LSR II (Becton Dickinson, San Diego, CA, USA) and FACSDIVA acquisition/analysis software (Becton Dickinson, San Diego, CA, USA) was used for data analysis.
4
Multiparametric Flow Cytometry for Stem Cell Subsets
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For staining of SKL cells (Sca-1+ c-Kit+ Lin–), VSELs (Sca-1+ Lin– CD45–), MSCs (Lin– CD45– CD31– CD90+), and EPCs (Lin– CD45– CD31+) the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–antimouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57–597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), conjugated with PE; anti-CD31 (clone MEC 13.3), conjugated with APC; and anti-CD90.2 (clone 30-H12), conjugated with BV510, were purchased from BD Biosciences. Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with an LSR II flow cytometer (BD Biosciences) [15 (link), 17 (link)].
5
Evaluating T-cell-mediated Cytotoxicity on 5T4-negative Tumor Cells
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MDA-MB-231 parental tumor cells and purified T cells were incubated with 10 μg/ml of DuoBody-CD3x5T4 or bsIgG1-CD3xctrl for 72 h. As a positive control for T-cell activation, T cells were stimulated with anti-CD3 (plates were pre-coated with 2 μg/ml anti-CD3 antibody [16-0037-85; eBioscience] at 37°C for 4 h) and anti-CD28 antibody (2 μg/ml, 16-0289-085; eBioscience) at 37°C for 72 h. Supernatant was transferred either directly (i.e., containing T cells and antibodies) or after pelleting of the T cells by centrifugation (i.e., cell-free supernatant) to MDA-MB-231 5T4 KO cells seeded in a 96-well flat-bottom plate 16 h prior. The MDA-MB-231 5T4 KO tumor cells were incubated with these supernatants or with freshly added T cells and antibody at 37°C for 72 h. After 72 h, supernatant and adherent cells (after trypsinization) were collected and analyzed for cytotoxicity (using the viability dye BV510 [564406; BD]) or Fas expression (FITC-labeled mouse antihuman Fas, 555673; BD Biosciences) by flow cytometry.
6
Multiparametric B Cell Analysis
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Briefly, single-cell suspensions from bone marrow, spleen, and peritoneal cavity lavage were isolated from immunized cLCM and, for comparison, C57BL/6 littermates. A total of 106 cells were suspended in FACS buffer (1× PBS (pH 7.2), 2% FBS, 2 mM EDTA) and B cells were stained with premixed combinations of fluorochrome-labeled mAbs at concentrations optimized by titration, and total B cells were gated as singlet, live, CD19+, and/or B220+ lymphocytes. All Abs were obtained from Biolegend unless otherwise stated. The primary labeled mAbs used were AF700 or AF594 conjugated α-B220, BV510 or APC-Cy7 α-CD19 (BD Biosciences, Franklin Lakes, NJ, USA), BV605-conjugated α-IgD, Percp cy5.5-conjugated α-IgM (BD Biosciences), PE-Cy7 conjugated α-CD21, APC-Cy7-labeled α-CD23, PE Cy7-conjugated α-CD93, PE Cy7-conjugated α-CD43, AF700-labeled CD5, FITC-conjugated α-kappa, and APC-conjugated α-lambda. 4′,6-diamidino-2-phenylindole (DAPI) was used to exclude dead cells. FACS was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and data was analyzed using FlowJo v10 (Tree Star, Ashland, OR, USA) software.
7
Flow Cytometry Analysis of B and NK Cells
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Samples of the spleen and the blood were collected for the flow cytometry from anesthetized mice at 3 d post B-cell depletion, 24 d after B-cell depletion/ 21 d (3 w) after AAV-PHP.eB injection, and 4 d of the NK-cell depletion. While the blood sample was simply collected, the removed spleen was cut into pieces and ground. Then, the samples were passed through 70 microns (um) nylon mesh for single-cell suspensions, followed by centrifugation at 300 × g for 10 min. Red blood cells in the sample were lysed with cell lysis buffer (559759, BD Pharmingen, San Jose, CA, USA), and the remaining cells were washed three times and counted (1×106 cells in a 100 µl for staining).
For B-cell staining, the antigens on the cell surface were labeled with anti-B220 (1 µg/100 µl, APC, BD Biosciences, San Jose, CA, USA), anti-CD3 (1 µg/100 µl, BV510, BD Biosciences), and anti-CD45 (1 µg/100 µl, Percp-cy5.5, BD Biosciences) at RT for 50 min. For NK-cell staining, cells were incubated with anti-NK49b (1 µg/100 µl, FITC, BD Biosciences), anti-CD3, and anti-CD45. Stained cells were collected with BD Verse (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
For B-cell staining, the antigens on the cell surface were labeled with anti-B220 (1 µg/100 µl, APC, BD Biosciences, San Jose, CA, USA), anti-CD3 (1 µg/100 µl, BV510, BD Biosciences), and anti-CD45 (1 µg/100 µl, Percp-cy5.5, BD Biosciences) at RT for 50 min. For NK-cell staining, cells were incubated with anti-NK49b (1 µg/100 µl, FITC, BD Biosciences), anti-CD3, and anti-CD45. Stained cells were collected with BD Verse (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
8
Reagent Device with Distinct Dried Dye Compositions
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Example 1
Experiments were performed to produce and test a reagent device having three distinctly positioned dried polymeric dye compositions according to embodiments of the present disclosure.
A first polymeric dye composition (BV510, a polymeric dye composition having an excitation maximum at 405 nm and an emission maximum at 510 nm; BD Biosciences, New Jersey) was positioned at a first location on an inner surface of a vial and dried. A second polymeric dye composition (BV421, a polymeric dye composition having an excitation maximum at 407 nm and an emission maximum at 421 nm; BD Biosciences, New Jersey) was distinctly positioned at a second separate location on the inner surface of the vial and dried. A third polymeric dye composition (BV605, a polymeric dye composition that includes a tandem fluorochrome that is a combination of BV421 and Cy™ 3.5 having an excitation maximum at 407 nm and an emission maximum at 602 nm; BD Biosciences, New Jersey) was distinctly positioned at a third separate location on the inner surface of the vial and dried. Cy™ 3.5 is a cyanine dye that can be excited by green (532 nm) and yellow-green (561 nm) lasers. A sample was added to the vial to produce a reconstituted dye composition and analyzed by flow cytometry. Graphs of the assay results are shown in
9
Phenotyping Antigen-Specific T Cells
10
Immunostaining of TNBC Cell Lines
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Individual TNBC cell lines were digested before immunofluorescence staining antibody treatment. Surface RON was detected using Zt/g4 after combination with anti-mouse IgG conjugated with Alexa Fluor 488 (BD, New York, NY); MET was detected using anti-MET mAb conjugated with BV510 (BD). Normal mouse IgG was used as the isotype control. All flow cytometry experiments were performed using BD FACSCanto II (BD).
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