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Fluorochrome conjugated mabs

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Fluorochrome-conjugated monoclonal antibodies (mAbs) are laboratory reagents that consist of a monoclonal antibody covalently linked to a fluorescent dye, known as a fluorochrome. These reagents are used in various flow cytometry and fluorescence-based applications to detect and quantify specific target molecules or cell populations.

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Lab products found in correlation

Cellquest software, by BD (4 mentions) Facscalibur flow cytometer, by BD (3 mentions) Lsrii analyzer, by BD (2 mentions) Intracellular staining kit, by BD (2 mentions) Golgiplug, by BD (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Isotype controls, by Miltenyi Biotec (1 mentions) Anti hla dr, by Miltenyi Biotec (1 mentions) Anti cd80, by Miltenyi Biotec (1 mentions) Anti cd83, by Miltenyi Biotec (1 mentions) Facscalibur, by BD (1 mentions) Anti cd86, by Miltenyi Biotec (1 mentions) Fluorochrome conjugated mabs, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions) Live dead fixable violet, by Thermo Fisher Scientific (1 mentions) Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)

19 protocols using fluorochrome conjugated mabs

1

Quantifying B Cell Cytokine Production

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Fluorochrome-conjugated mAbs were from BD Biosciences, eBioscience or Biolegend. α-TIM-1-PE (RMT1-4; Biolegend) whose binding does not overlap with RMT1-10, was used to determine TIM-1 expression(5 (link)). All staining was performed in the presence of Fc block. Data was acquired on LSRII analyzers (BD), and analyzed using FlowJo software (TreeStar). Background staining was determined with isotype-matched controls. For detection of intracellular cytokines, B cells were cultured for 5 hours with LPS, PMA, ionomycin, and monensin(5 (link)). Leukocytes from IL-4-/-, IL-10−/− mice served as negative control to demonstrate specificity and background-staining.
Yeung M.Y., Ding Q., Brooks C.R., Xiao S., Workman C.J., Vignali D.A., Ueno T., Padera R.F., Kuchroo V.K., Najafian N, & Rothstein D.M. (2015). TIM-1 signaling is required for Maintenance and Induction of regulatory B cells. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(4), 942-953.
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2

Tfh-like Cells and Plasmablasts in AS

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The frequency and phenotype of Tfh-like cells and of plasmablasts present in the peripheral blood of AS patients and HC was assessed by flow cytometry after staining freshly isolated PBMCs with antibodies directed against surface phenotypical markers. Fluorochrome-conjugated mAbs from BD Pharmingen (San Diego, CA, USA) were used to examine the expression of CD3, CD4, CD8, CXCR5, ICOS, PD-1, CCR6, CXCR3, CD19, CD20, CD27 and CD38 in a FACSCalibur flow cytometer with CellQuest software (BD Biosciences).
Bautista-Caro M.B., Arroyo-Villa I., Castillo-Gallego C., de Miguel E., Peiteado D., Plasencia-Rodríguez C., Villalba A., Sánchez-Mateos P., Puig-Kröger A., Martín-Mola E, & Miranda-Carús M.E. (2014). Decreased Frequencies of Circulating Follicular Helper T Cell Counterparts and Plasmablasts in Ankylosing Spondylitis Patients Naïve for TNF Blockers. PLoS ONE, 9(9), e107086.
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3

Profiling Tfh-like Cells and Plasmablasts in RA

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The frequency and phenotype of Tfh-like cells and plasmablasts present in the peripheral blood of RA patients and HC was assessed by flow cytometry in a FACSCalibur flow cytometer using CellQuest software (BD Biosciences), after staining freshly isolated PBMCs with antibodies directed against surface phenotypic markers. Fluorochrome-conjugated mAbs from BD Pharmingen (San Diego, CA, USA) were used to examine the expression of CD3, CD4, CD8, CXCR5, ICOS, PD-1, CCR6, CXCR3, CD19, CD20, and CD38.
Arroyo-Villa I., Bautista-Caro M.B., Balsa A., Aguado-Acín P., Bonilla-Hernán M.G., Plasencia C., Villalba A., Nuño L., Puig-Kröger A., Martín-Mola E, & Miranda-Carús M.E. (2014). Constitutively altered frequencies of circulating follicullar helper T cell counterparts and their subsets in rheumatoid arthritis. Arthritis Research & Therapy, 16(6), 500.
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4

Characterization of Dendritic Cells

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For analysis of DC, the following monoclonal antibodies were used: anti-CD14, anti-HLA-DR, anti-CD80, anti-CD86, anti-CD83, and isotype controls (all from Miltenyi). 1 × 105 cells/tube were stained with fluorochrome conjugated mAbs (BD) and incubated for 30 min at 4 °C in the dark. All labeled cells were analyzed on a FACS Calibur (BD). Data analyses were conducted using CellQuest software (Becton Dickinson Company, Franklin Lakes, NJ, USA).
Nava S., Lisini D., Frigerio S., Pogliani S., Pellegatta S., Gatti L., Finocchiaro G., Bersano A, & Parati E.A. (2020). PGE2 Is Crucial for the Generation of FAST Whole- Tumor-Antigens Loaded Dendritic Cells Suitable for Immunotherapy in Glioblastoma. Pharmaceutics, 12(3), 215.
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5

Breg Cell Phenotyping in Ankylosing Spondylitis

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The frequency and phenotype of Breg cells present in the peripheral blood of AS patients and HC was assessed by flow cytometry after staining freshly isolated PBMCs with antibodies directed to surface phenotypical markers. Fluorochrome-conjugated mAbs from BD Pharmingen (San Diego, CA, USA) were used to examine the expression of CD3, CD4, CD19, CD24 and CD38 in a FACSCalibur flow cytometer with CellQuest software (BD Biosciences).
Bautista-Caro M.B., de Miguel E., Peiteado D., Plasencia-Rodríguez C., Villalba A., Monjo-Henry I., Puig-Kröger A., Sánchez-Mateos P., Martín-Mola E, & Miranda-Carús M.E. (2017). Increased frequency of circulating CD19+CD24hiCD38hi B cells with regulatory capacity in patients with Ankylosing spondylitis (AS) naïve for biological agents. PLoS ONE, 12(7), e0180726.
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6

Cell Surface Marker and Cytokine Analysis

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For cell surface marker analysis, we stained cell samples with fluorochrome-conjugated mAbs (BD Biosciences, San Jose, CA, USA) for 30 min, then washed the samples three times with 2% FBS in PBS. For intracellular cytokine or transcription factor analysis, cells were stimulated with 50 ng/ml phorbol myristate acetate plus 500 ng/ml ionomycin for 4 h in the presence of 1 µg/ml brefeldin A (BD Biosciences, San Jose, CA, USA), then fixed and permeabilized, followed by cytoplasmic staining with the appropriate fluorochrome-conjugated mAbs (eBioscience, San Diego, CA, USA). We performed sample acquisition with a FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed the results using FlowJo software.
Yang F., Fan X., Huang H., Dang Q., Lei H, & Li Y. (2018). A Single Microorganism Epitope Attenuates the Development of Murine Autoimmune Arthritis: Regulation of Dendritic Cells via the Mannose Receptor. Frontiers in Immunology, 9, 1528.
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7

Phenotypic Analysis of G-PBSC Cell Products

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Phenotypic characterization of the initial G-PBSC and CD45RA-depleted G-PBSC cell products was performed using a custom LSR II flow cytometer equipped with 405 nm, 488 nm, 532 nm and 635 nm lasers (BD Biosciences). Fluorochrome conjugated mAbs to the following molecules were obtained from BD Biosciences: CD3, CD8, CD4, CD45RO, CD45RA, CD27, CD28, CD19, CD56, CD16, CD14, CD34, IL2 and IFN-γ; antibodies to CD25 and FoxP3 were from eBioscience (San Diego, CA), CCR7 from R & D Systems (Minneapolis, MN), and CD28 from Beckman Coulter (Indianapolis, IN). MHC-tetramer analysis for viral-specific T cells was performed using iTag MHC tetramers purchased from Beckman Coulter. G-PBSC were surface labeled with antibodies or tetramers for 20 or 30 minutes respectively at 4°C. For T regulatory cell analysis, samples were fixed in Fixation Permeabilization solution before washing and staining with anti-FoxP3 mAb in 1X Permeabilization buffer (e-Bioscience). Dead-cell exclusion was performed using either propidium iodide/RNAse staining buffer (BD Biosciences), DAPI (4’6-diamidino-2 phenylindole, Sigma-Aldrich Saint Louis, MO) or Live/Dead Fixable Violet (Molecular Probes, Eugene, OR). Analysis was performed using FlowJo software (Treestar, Ashland, OR).
Bleakley M., Heimfeld S., Jones L.A., Turtle C., Riddell S.R, & Shlomchik W. (2014). Engineering Human Peripheral Blood Stem Cell Grafts That Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T cells. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(5), 705-716.
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8

Multiparametric Flow Cytometry Analysis

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Surface staining and intracellular cytokine staining were performed as previously described.24 (link) Briefly, surface staining was performed in 100 µl PBS with 3% (v/v) FBS and different antibody cocktails for surface markers (anti-CD3e, anti-CD4, anti-CD8a, anti-CD44, anti-CD62L, anti-B220, anti-CD11b, anti-Ly6C, anti-Ly6G and anti-CD11c) at room temperature for 30 min. Post-staining for surface markers (anti-CD3e, anti-CD4, anti-CD8a and anti-CD44), the intracellular cytokines were stained using intracellular staining kits from BD Biosciences. The cells were incubated with anti-IFN-γ, anti-IL-2 and anti-TNF-α for 40 min at room temperature. OVA-specific CD8 T cells were also quantified by direct staining with H2-Kb/OVA257–264 (MHC/peptide) tetramers. All samples were analyzed on a FACS Canto II flow cytometer (BD Biosciences), and all fluorochrome-conjugated mAbs were purchased from BD Pharmingen (San Jose, CA, USA).
Zeng W., Wang Y., Liu Z., Khanniche A., Hu Q., Feng Y., Ye W., Yang J., Wang S., Zhou L., Shen H, & Wang Y. (2014). Long-term exposure to decabrominated diphenyl ether impairs CD8 T-cell function in adult mice. Cellular and Molecular Immunology, 11(4), 367-376.
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9

Monocyte Phenotyping with HLA-DR and CD86

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Monocytes were detached with 4 mM EDTA for 1 h, washed, and resuspended in phosphate buffered saline (PBS) supplemented with 1% heat-inactivated fetal bovine serum. The monocytes were then stained with fluorochrome-conjugated mAbs for human leukocyte antigen-DR isotype (HLA-DR) and CD86 (BD science, Erembodegem-Dorp, Belgium) or control Abs. Data was acquired on a Cytoflex cytometer (Beckman Coulter, Kraemer Boulevard Brea, CA, USA) and analyzed using FlowJo 10.3 software.
Xu Y., Huang Z., Yu X., Li Z., Zheng L, & Xu J. (2021). HHLA2 Expression is Associated with Poor Survival in Patients with Hepatocellular Carcinoma. Biologics : Targets & Therapy, 15, 329-341.
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10

Multicolor Flow Cytometry for Immune Cell Analysis

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Flow cytometry was carried out using distinct fluorochrome-conjugated mAbs that recognize human CD3, CD4, and CD8 (BD Biosciences), human TCRα, TCRβ (BioLegend). These mAbs were labeled with Alexa Fluor 488, phycoerythrin (PE), phycoerythrin–cyanine 5 (PC5), phycoerythrin-cyanin 7 (PC7) or allophycocyanine. The cells were examined by means of multicolor flow cytometry on the BD FACSVerse flow cytometer (BD Biosciences), and data were analyzed with the Flowjo™ V10 software (BD Biosciences).
Jiang H., Wang C.W., Wang Z., Dai Y., Zhu Y., Lee Y.S., Cao Y., Chung W.H., Ouyang S, & Wang H. (2022). Functional and structural characteristics of HLA-B*13:01-mediated specific T cells reaction in dapsone-induced drug hypersensitivity. Journal of Biomedical Science, 29, 58.
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