One hundred thousand cells were incubated with three distinct panels with fluorescent-labeled antibodies: panel 1 for progenitor cell subtypes PE-MSCA1, PeVio770-CD14, APC-CD271 (Miltenyi Biotec, Paris, France); PerCP-CD34, V450-CD31, BV510-CD45 (BD Biosciences, Le Pont De Claix, France); panel 2 for macrophage subtypes: PeVio770-CD14, APC-CD206, BV510-CD45 (BD Biosciences); panel 3 for lymphocytes FITC-CD4, PerCP-CD8, Pe-Cy7-CD56; APC-Cy7-CD19, V450-CD3; BV510-CD45 (BD Biosciences, Le Pont De Claix, France) or an isotype control panel for 30 min at 4 °C in phosphate buffered saline (PBS, Eurobio Scientific, Les Ulis, France) supplemented with 0.5% bovine serum albumin (BSA, Gibco, ThermoFisher Scientific, Les Ulis, France) and 2 mmol/L ethylenediaminetetraacetic acid (EDTA). Cells were washed with PBS and analyzed using a FACS CantoTM II flow cytometer and Diva Pro software (BD Biosciences, Le Pont De Claix, France). Cell subsets were identified through the combination of cell surface markers as described in Table 2 and the gating strategy shown in Figure 1 B.
Cd45 bv510
Manufactured by BD
Sourced in United States, France
CD45-BV510 is a fluorescently-labeled antibody that binds to the CD45 antigen, which is expressed on the surface of various hematopoietic cells. This product is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (3 mentions)
Igd fitc, by BD (2 mentions)
Cd138 bv421, by BD (2 mentions)
Cd44 fitc, by BD (2 mentions)
Cd54 pe, by BD (2 mentions)
Anti human cd133 apc, by BD (2 mentions)
Cd54 percp cy5, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Prism 5, by GraphPad (2 mentions)
Cd11b fitc, by BD (2 mentions)
Cd11c pe cy7, by BD (2 mentions)
Ly6g alexa fluor 700, by BD (2 mentions)
Lsrfortessa x 20 cell analyzer, by BD (2 mentions)
Ly6c percp cy5, by BD (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Dnase, by Merck Group (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Cd8 alexa fluor 700, by BD (2 mentions)
Pd l1 pe, by BD (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
29 protocols using cd45 bv510
1
Multiparameter Flow Cytometry Analysis of Cell Subsets
2
Immunophenotyping of T and B Cells
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After isolation, PBMCs were stained with the following fluorochrome-conjugated antibodies: CXCR3 (CD183) FITC, CCR6 PE-Vio770 (Miltenyi Biotec, Paris, France); CXCR5 (CD185) PE (eBioscience, ThermoFisher, Villebon, France); CD45RA ECD, PD-1 (CD279) PECY5.5, ICOS (CD278) APC, CD3 AA750 (Beckman Coulter, Villepinte, France); CD45 BV510, CD4 BV650, HLA-DR BV786 (BD Biosciences, Rungis, France). For B-lymphocyte analysis, the following fluorochrome-conjugated antibodies were used: IgD FITC, CD21 BV450, CD45 BV510, IL-21R APC (BD Biosciences); CD27 PE, CD24 ECD, CD38 PECY5.5, CD19 PC7 (Beckman Coulter); CD5 AA700 (Biolegend, Ozyme, Montigny-le-Bretonneux). T-lymphocyte and B-lymphocyte absolute numbers were determined using 50 µL of whole blood from patients and HC using Trucount tubes (BD Biosciences). Samples were stained with the following antibodies: CD19 FITC, CD45 ECD, CD3 AA750 (Beckman Coulter) for 15 min and then incubated for 15 min with 450 µL BD FACS Lysis Solution and subsequently analysed by flow cytometry. Compensation beads were used for compensation setting (VersaComp; Beckman Coulter). Cells were analysed on a Cytoflex flow cytometer (Beckman Coulter). Data were analysed using Kaluza V.5.1 software (Beckman Coulter).
3
Multiparametric Flow Cytometry Immunophenotyping
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Cell surface staining was processed using whole blood lysis method. Freshly drawn heparinized peripheral blood samples were labeled with monoclonal antibody (mAb) panel for T lymphocyte subsets [anti-CD3-AlexaFlour700, -CD4-APC, -CD8-APC/CY7, -CD45-BV510, -CD45RA-FITC, -CD45RO-PE, -CCR7-PE and -HLA-DR-BV711 (all from Biolegend, San Jose, CA, USA)], mAb panel for B lymphocyte subsets [anti-CD19-APC, -CD24-PE, -CD27-PE/CY7, -CD38-AlexaFlour700, -CD45-BV510, -CD138-BV421 and -IgD-FITC (all from BD-Biosciences, San Jose, CA, USA)], and with mAb panel for Treg [anti-CD3-AlexaFlour700, -CD4-APC, -CD25-PE, -CD45-BV510, -CD127-BV421 (all from BD-Biosciences, San Jose, CA, USA)]. Following incubation, erythrocytes were lysed using FACS Lysing Solution (BD Biosciences, San Jose, CA, USA), and at least 10.000 cells were collected in CD45+ lymphocyte gate. The data were acquired on a NovoCyte flow cytometer (Agilent Technologies, USA) and analyzed using the NovoExpress operating system software (Agilent Technologies, USA).
4
Multicolor Flow Cytometry of Tumor Cells
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Tumor cells were resuspended at a concentration of 106 cells/100 µL and cell surface antigens were stained with fluorophore-conjugated antibodies: NIR-L/D (Invitrogen, Waltham, MA, USA, cat. 17-5321-81), BV510-CD45 (BD, Franklin Lakes, NJ, USA, cat. 563891), PE-Cy7-F4/80 (Invitrogen, cat. 25-4801-82), FITC-CD206 (BioLegend, San Diego, CA, USA, cat. 141704), APC-CD3 (eBioscience, San Diego, CA, USA, cat. 17-0031-82), FITC-CD4 (Thermo Fisher, Waltham, MA, USA, cat. 11-0041-82), PE-Cy5.5-CD8a (ThermoFisher, cat. 45-0081-42), and PE-CD25 (Invitrogen, cat. 12-0251-83). Antibody staining occurred for 30 min at 4 °C in the dark. Cells were acquired on the Attune NxT flow cytometer (Thermo Fisher). Compensation and sequential gating (see Supplementary Materials ) were performed with FlowJo software (FlowJo LLC, v.10.8.1; Ashland, OR, USA).
5
Whole Blood Cytokine Secretion Assay
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Whole
blood stimulations were performed as described above, except that
cells were stimulated for 2 h 37 °C, 5% CO2, followed
by 4 h stimulation in the presence of Brefeldin A (BD Bioscience)
and Monensin (BD Bioscience) according to manufacturer’s recommendations
to block cytokine secretion. Subsequently, red blood cell lysis is
performed as described above and cells are stained with LIVE/DEAD
Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according to manufacturer’s
recommendations. To allow intracellular cytokine staining, the cells
are treated with Cytofix/Cytoperm kit (BD Bioscience) according to
manufacturer’s recommendations. The following markers were
used to identify cell subsets: CD45-BV510 (BD Bioscience), CD3-APC-Cy7
(BD Bioscience), CD14-PeCy7 (Biolegend), and CD66b-BV421 (BD Bioscience).
Each sample divided was separately analyzed for the presence of IL-6
(IL-6-PECF594, Clone: MQ2-13A5, BD Bioscience), IL-8 (IL-8-PECF594,
clone: G265–8, BD Bioscience), or TNF-α (TNF-α-PECF594,
clone: MAB11, BD Bioscience). Flow cytometry data were collected on
a BD LSR II and analyzed using FlowJo 10.2 Software (Figure S2 ). Dead cells and CD45 negative events were excluded
from the data analysis.
blood stimulations were performed as described above, except that
cells were stimulated for 2 h 37 °C, 5% CO2, followed
by 4 h stimulation in the presence of Brefeldin A (BD Bioscience)
and Monensin (BD Bioscience) according to manufacturer’s recommendations
to block cytokine secretion. Subsequently, red blood cell lysis is
performed as described above and cells are stained with LIVE/DEAD
Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according to manufacturer’s
recommendations. To allow intracellular cytokine staining, the cells
are treated with Cytofix/Cytoperm kit (BD Bioscience) according to
manufacturer’s recommendations. The following markers were
used to identify cell subsets: CD45-BV510 (BD Bioscience), CD3-APC-Cy7
(BD Bioscience), CD14-PeCy7 (Biolegend), and CD66b-BV421 (BD Bioscience).
Each sample divided was separately analyzed for the presence of IL-6
(IL-6-PECF594, Clone: MQ2-13A5, BD Bioscience), IL-8 (IL-8-PECF594,
clone: G265–8, BD Bioscience), or TNF-α (TNF-α-PECF594,
clone: MAB11, BD Bioscience). Flow cytometry data were collected on
a BD LSR II and analyzed using FlowJo 10.2 Software (
from the data analysis.
6
Immunofluorescence and Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
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For immunofluorescence studies, tumors were fixed for 24 h in 4% paraformaldehyde (PFA), followed by overnight incubation in a 30% sucrose solution. Tumors were then snap-frozen in optimal cutting temperature compound (OCT) and 5 μm sections were obtained. For CD8 (Cell Signaling, Danvers, MA, USA, 98941) and CD11c (Cell Signaling, 97585S) staining, slides were fixed for 10 min in 4% PFA before antigen retrieval in citrate buffer (pH 5.7) with 0.5% Triton. After the addition of an AlexaFluor-488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA, A11034), slides were scanned (3DHistech Pannoramic P250 Flash III, Budapest, Hungary, 20X) and the extent of CD8 and CD11c staining was quantified over the total tumor area using Visiopharm software. For TIL analysis by flow cytometry, tumors were mechanically dissociated and single cell suspension was obtained by passing them through a 70 µm strainer. Cells were stained with CD45-BV510 (BD Pharm, 513151), CD3-APC (BD Pharm, 553066), CD8a-FITC (BD Pharm, 553031) and CD4-BV421 (BD Pharm, 553066) for 15 min RT.
7
Characterizing CTCs in Colorectal Cancer
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Peripheral blood samples were obtained from CRC patients attending our department and an informed consent was obtained from all the individuals. Peripheral blood samples were collected and prepared as per the protocol described in our previous report 19 . In detail, CTCs from cell suspensions were characterized by multiparameter flow cytometry. The antibodies used in this study included anti‐human CD133‐APC, CD44‐FITC, CD54‐PErcp‐cy5.5, CD54‐PE, and CD45‐BV510 (all antibodies were purchased from BD Biosciences, San Diego, CA, USA). DAPI was used to identify and sort the dead cells. The remaining steps were the same as the protocol described in our previous report 19 . The absolute CTCs or antibody‐positive cell numbers were derived from the absolute number of white blood cells provided by the hematological analyzer, and the percentage of CTCs or antibody‐positive cells was determined by flow cytometry, using the following formula: percentage of cells × white blood cells count/100.
8
Multicolor Flow Cytometry for Hematopoietic Cells
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For flow-cytometric analysis hematopoietic cells and ECs were stained with different combinations of monoclonal fluorochrome-conjugated antibodies (see Supplemental Table 5 ) for at least 20 min at 4 °C. Propidium-Iodide (PI) or 7-Aminoactinomycin D (7-AAD) were used for dead cell exclusion. Appropriate isotype-matched, control monoclonal antibodies were used to determine the level of background staining in all experiments. Flow cytometric analyses were performed on a FC500 flow cytometer equipped with the CXP 2.2 software (Beckman Coulter) (Fig. 1C ). Cells were sorted using a FACSAria I cell sorter. The sort-purity was routinely assessed by recovery of sorted cells and was >99.5%.
Samples from murine BM were stained with the following fluorochrome conjugated antibodies: anti-CD34-APC-AF750 (Beckman Coulter), anti-CD133-APC (Miltenyi Biotec), anti-CD45RA-BV711 (BioLegend), anti-CD38-BV785 (BD Biosciences), anti-CD10-PeCF594 (BD Biosciences) and CD45-BV510 (BD Biosciences). DAPI staining was used for excluding dead cells. All measurements were performed in a BD FACSAriaTMIII (Beckman Coulter) flow cytometer.
Samples from murine BM were stained with the following fluorochrome conjugated antibodies: anti-CD34-APC-AF750 (Beckman Coulter), anti-CD133-APC (Miltenyi Biotec), anti-CD45RA-BV711 (BioLegend), anti-CD38-BV785 (BD Biosciences), anti-CD10-PeCF594 (BD Biosciences) and CD45-BV510 (BD Biosciences). DAPI staining was used for excluding dead cells. All measurements were performed in a BD FACSAriaTMIII (Beckman Coulter) flow cytometer.
9
Multiparameter Flow Cytometry of CTCs
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CTCs from cells suspension were characterized by multiparameter flow cytometry. The antibodies used in this study include: anti-human CD133-APC, CD44-FITC, CD44-APC-Cy7, CD54-PErcp-cy5.5, CD54-PE, CD24-PE/Cy7, CD10-PECF594, CD26-PE, CD166-Percp-cy5.5, CD45-BV510, CD58-PE, CD66-PE, CD71-PE, CD117-PE, EPCAM-Percp-cy5.5, and EGFR-PE (all of the above-mentioned antibodies were purchased from BD Biosciences). DAPI was used to identify the dead cells. Evaluation of nucleated cells from whole cells suspensions was carried out using a FACS Canto Flow Cytometer (BD Biosciences) and data were analyzed using BD FACS Diva software. A range of internal quality assurance procedures was employed, including daily calibration of the optical alignment and fluidic stability of the flow cytometer using the seven-color Set-up Beads (BD Biosciences). The absolute CTCs or antibody-positive cell number was derived from the absolute number of the white blood cells provided by the hematological analyzer and percentage of CTCs or antibody-positive cell as determined by flow cytometry, using the following formula: percentage of cells × white blood cells count/100.
10
Multicolor Flow Cytometry Analysis
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We performed FACS analysis of CD11b, CD14 and CD15 expression using the following anti-human monoclonal antibodies: CD11b-PE (Beckman Coulter, IM2581U), CD14-AF700 (BD, 557923), CD15-APC (BD, 551376), CD45-BV510 (BD, 563204) or CD45-APCH7 (BD, 641417). Apoptotic cells were stained with AnnexinV-BV421 (BD, 563973) or Annexin-FITC (BD, 556420) according to the manufacturer’s protocol (BD).
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