Fluorochrome-labeled monoclonal antibodies recognizing CD1a, CD14, CD16, CD83, CD80, CD86, were obtained from Becton Dickinson (Allschwil, Switzerland); CRT, HSP70- and HSP90-specific labeled antibodies were obtained from Abcam (Cambridge, United Kingdom). After treatment with the decoction, cells were collected with TryPLE Express (Thermo Fisher Scientific) and stained for 20 min with the appropriate antibody. Specific binding was evaluated by flow cytometry [FACScalibur (Becton Dickinson); Guava EasyCyte 6-2L (Merck, Darmstadt, Germany); CytoFLEX (Beckman Coulter, Brea, CA, USA)]. PI or Sytox Red (Thermo Fisher Scientific) were added to each analyzed sample to discriminate live and dead cells. The permeabilized cells were excluded from DAMPs analysis, and the fold changes in the MFI were analyzed.
Fluorochrome labeled monoclonal antibodies
Manufactured by BD
Sourced in United States
Fluorochrome-labeled monoclonal antibodies are laboratory reagents used in various immunoassay techniques. They consist of a monoclonal antibody that is chemically coupled to a fluorescent dye or fluorochrome. These labeled antibodies are used to detect and quantify specific target molecules or cells in samples, such as in flow cytometry, immunohistochemistry, and fluorescence microscopy applications.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Sytox red, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Guava easycyte 6 2l, by Merck Group (1 mentions)
Cellquest, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Lsrii cell analyzer, by BD (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
Igg isotype control, by BD (1 mentions)
Facs caliber, by BD (1 mentions)
Facsdiva 8, by BD (1 mentions)
Live dead cell stain, by Thermo Fisher Scientific (1 mentions)
Calibrite beads, by BD (1 mentions)
Apc cy7, by BD (1 mentions)
8 protocols using fluorochrome labeled monoclonal antibodies
1
Phenotyping Immune Cell Populations
2
PBMC Immunophenotyping with V(H)1-69 Antibody
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Peripheral blood mononuclear cells (PBMCs) were obtained by density-gradient centrifugation; for some experiments B cells were purified by negative selection with immunomagnetic beads. Immunophenotyping was done using combinations of fluorochrome-labeled monoclonal antibodies (all from Becton-Dickinson Biosciences). The G6 monoclonal antibody, which recognizes an epitope of the VH1-69-encoded protein, was kindly provided by R. Jefferis, Birmingham, UK. Unlabeled G6 was counterstained with FITC- or PE-conjugated goat anti-mouse IgG (Becton-Dickinson Biosciences) using mouse IgG as control as previously described (11 (link), 16 (link), 17 (link)). Flow cytometric analyses were done on a FACSCalibur instrument (Becton-Dickinson Biosciences) using the CellQuest (Becton-Dickinson Biosciences) and FlowJo (Tree Star, Ashland, OR) software.
3
Immunophenotyping of Placental Cell Populations
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The method #1 and #2 were used for obtaining cells from native and cryopreserved placental samples for flow cytometry. For immunophenotyping of cells from tissues studied (FTPT, FiTPT, CB, and FL), we used the following fluorochrome-labeled monoclonal antibodies (Becton Dickinson, USA): anti-CD34 APC, anti-CD90 FITC, anti-CD45 APC-Cy7, anti-CD14 Pacific Blue, anti-CD31 PE, anti-CD133 PE, anti-CD45RA FITC, anti-CD7 PE, anti-CD19 PE-Cy7, anti-CD33 FITC, and anti-CD235a PE. The analysis was performed only for cell populations excluding 7-AAD dye. Immunophenotyping was performed on a cell sorter BD FACSAria (Becton Dickinson, USA) using the BD FACS Diva 6.1.2 software and analyzing simultaneously 2 parameters of light scattering and 7 parameters of fluorescence. Control samples were used to adjust the compensation of fluorochrome overlap: unstained, single stained, fluorescence minus one, and isotype controls. “Expression level” means the percentage of cells expressing a particular antigen. “Intensity of expression” represents the intensity of expression of a particular antigen by a specific cell population.
4
Flow Cytometric Lymphocyte Analysis
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Immunophenotyping for B and T lymphocytes was performed with flowcytometry post-haploHSCT. Briefly, fresh peripheral blood cells were stained with fluorochrome-labeled monoclonal antibodies against cluster of differentiation (CD) cell surface molecules, CD19, CD3, CD4, CD8, CD45RO, CD45RA, and CD28 (Beckton Dickinson, San Jose, CA, USA). Polychromatic flow cytometric analyses were performed with a custom 7-laser high resolution LSRII cell analyzer (Beckton Dickinson).
5
Peripheral Blood Immunophenotyping in Healthy Controls
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Immunophenotyping of peripheral blood samples at baseline and after 12‐month follow‐up was performed with combinations of fluorochrome‐labeled monoclonal antibodies (Becton‐Dickinson Biosciences, San Jose, CA, USA) to CD19‐PC5.5, CD21‐phycoerythrin (PE), CD27‐allophycocyanin (APC) and immunoglobulin (Ig)D‐fluorescein isothiocyanate (FITC). Flow cytometry analysis was performed on a BD fluorescence activated cell sorter (FACS)Calibur system and data files were acquired and analysed using CELLQuest version 3.3 (Becton Dickinson, Mountain View, CA, USA) and FlowJo (TreeStar, Inc., Ashland, OR, USA) software. We chose a threshold of 10% for CD21low B cells. This value has been chosen as media ± 2 standard deviations (s.d.) of the CD21low B cells percentage of healthy controls, according to our previous study (4 (link)).
6
Comprehensive Immune Biomarker Analysis
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To determine IL-2 and IL-10 levels in the serum, animals were bled periodically through retro-orbital puncture. IL-2 and IL-10 serum levels were determined using commercial ELISA kit according to the manufactures instructions (Biolegend Inc, San Diego, CA, USA). For flow cytometric analysis mononuclear cells from blood and spleen of the animals were stained with appropriate fluorochrome-labeled monoclonal antibodies directed at human CD3, CD4, CD8 and CD45 (from BD Biosciences, San Diego, CA or Tonbo Biosciences, San Diego, CA). The beta mark TCR repertoire kit from Beckman Coulter was used for Human Vβ analysis. All flow cytometry data were acquired on FACS Canto II instrument (BD) and analyzed with FlowJo software. Serum levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ and TNF-α were measured using human Th1/Th2/Th17 and Inflammatory Cytokine Bead Array kits (BD Pharmingen, San Diego, CA) according to manufacturer's instructions.
7
FACS Analysis of Lung and Lymph Node Cells
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Enzymatic digestion was performed for fluorescence-activated cell sorting (FACS) analysis of cells in lung tissue and mesenteric lymph nodes (MLN). The lung and MLN were removed from the mouse, washed three times, cut, and moved to a tube with Hank’s Balanced Salt Solution including 2% FBS and 1 mM Ethylene-diamine-tetraacetic acid (EDTA) for 30 min. The lung tissue was reacted with 1 mg/mL collagenase IV, filtered through a cell strainer, and centrifuged for 20 min. Collected cells from lung and MLN and BALF were rinsed twice and incubated with Immunoglobulin G (IgG) antibodies against CD8 (53-6.7, rat IgG2a), CD4 (RM4-5, rat IgG2a), CD21 (7G6, rat IgG2b), CD69 (H1.2F3, Hamster IgG1), B220 (RA3-6B2, rat IgG2a), SiglecF (1RNM44N, rat IgG2a), CD62L (MEL-14, rat IgG2a), Gr-1 (RB6-8C5, rat IgG2b), CD11b (M1/70, rat IgG2b), and CD44 (IM7, rat IgG2b). All fluorochrome-labeled monoclonal antibodies and IgG isotype controls were obtained from BD Biosciences (San Diego, CA, USA). Cells from the lung and BALF were reacted with fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-labeled monoclonal antibodies for 30 min. The stained cells were characterized using FACS Caliber (BD Biosciences) according to a previously described method [27 (link)].
8
Multi-Color Flow Cytometry of Immune Cells
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Flow cytometry was performed on venous peripheral blood collected in heparin-coated vacutainer tubes using a FACSAria III 8-colour flow-cytometer (BD Biosciences, San Jose, CA, USA), daily calibrated with caliBRITE beads (Fitc, Pe, PerCP, and APC) and compbeads (Pe-Cy7 and APC-Cy7; BD Bioscience, San Jose, CA, USA). Fluorochrome-labeled monoclonal antibodies (BD Bioscience) for the identification of NK and T cells were Horizon-V450-anti-CD16, fluorescent isothiocyanate (FITC)-anti-CD8, Pe-Cy7-anti-CD56, and APC-Cy7-anti-CD3. Viability was analyzed using LIVE/DEAD cell stain (Invitrogen). Cells were stained with antibodies for 30 min at 4°C and washed with FACS buffer (PBS/0.2% BSA/0.01% NaN3). A minimum of 100,000 events for each sample were collected and analyzed using FACSDiva™ 8.0 Software (BD Bioscience).
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