Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens

2014 ◽  
pp. n/a-n/a ◽  
Author(s):  
Martina De Vita ◽  
Valentina Catzola ◽  
Alexia Buzzonetti ◽  
Marco Fossati ◽  
Alessandra Battaglia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Surface Staining ◽  
Surface Staining

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

scholarly journals Cyclopropene derivatives of aminosugars for metabolic glycoengineering

2019 ◽  
Vol 15 ◽  
pp. 584-601 ◽  
Author(s):  
Jessica Hassenrück ◽  
Valentin Wittmann
Keyword(s):  
Cell Surface ◽  
Diels Alder ◽  
Inverse Electron Demand ◽  
Distinct Cell ◽  
Chemical Reporter ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Metabolic Glycoengineering ◽  
Derivatives Of ◽  
Electron Demand

Cyclopropenes have been proven valuable chemical reporter groups for metabolic glycoengineering (MGE). They readily react with tetrazines in an inverse electron-demand Diels–Alder (DAinv) reaction, a prime example of a bioorthogonal ligation reaction, allowing their visualization in biological systems. Here, we present a comparative study of six cyclopropene-modified hexosamine derivatives and their suitability for MGE. Three mannosamine derivatives in which the cyclopropene moiety is attached to the sugar by either an amide or a carbamate linkage and that differ by the presence or absence of a stabilizing methyl group at the double bond have been examined. We determined their DAinv reaction kinetics and their labeling intensities after metabolic incorporation. To determine the efficiencies by which the derivatives are metabolized to sialic acids, we synthesized and investigated the corresponding cyclopropane derivatives because cyclopropenes are not stable under the analysis conditions. From these experiments, it became obvious that N-(cycloprop-2-en-1-ylcarbonyl)-modified (Cp-modified) mannosamine has the highest metabolic acceptance. However, carbamate-linked N-(2-methylcycloprop-2-en-1-ylmethyloxycarbonyl)-modified (Cyoc-modified) mannosamine despite its lower metabolic acceptance results in the same cell-surface labeling intensity due to its superior reactivity in the DAinv reaction. Based on the high incorporation efficiency of the Cp derivative we synthesized and investigated two new Cp-modified glucosamine and galactosamine derivatives. Both compounds lead to comparable, distinct cell-surface staining after MGE. We further found that the amide-linked Cp-modified glucosamine derivative but not the Cyoc-modified glucosamine is metabolically converted to the corresponding sialic acid.

scholarly journals Modeling Adenovirus Latency in Human Lymphocyte Cell Lines

2010 ◽  
Vol 84 (17) ◽  
pp. 8799-8810 ◽  
Author(s):  
Yange Zhang ◽  
Wen Huang ◽  
David A. Ornelles ◽  
Linda R. Gooding
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Virus Genome ◽  
Mrna Levels ◽  
Late Gene Expression ◽  
Retroviral Transduction ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Infected Cells ◽  
Adenovirus Receptor

ABSTRACT Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection.

scholarly journals STAINING OF BLOOD CELL SURFACES WITH A LECTIN-HORSERADISH PEROXIDASE METHOD

1973 ◽  
Vol 21 (10) ◽  
pp. 912-922 ◽  
Author(s):  
RICHARD T. PARMLEY ◽  
B. J. MARTIN ◽  
S. S. SPICER
Keyword(s):  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Blood Cell ◽  
Mononuclear Cells ◽  
Periodate Oxidation ◽  
Cell Surfaces ◽  
Con A ◽  
Bone Marrow Precursor ◽  
Cell Surface Staining ◽  
Surface Staining

Human and rabbit blood cell surfaces were stained by a lectin-horseradish peroxidase (HRP) technique using either concanavalin A (Con A) or Lens culinaris hemagglutinins (LcH). Intensity of staining of the human cells with the Con A-HRP method decreased in the order: platelets, mononuclear cells, granulocytes, erythrocytes. Cell surface staining of mature cells was similar to the bone marrow precursor cells as well as to leukemic blast cells. Surface staining of erythrocytes correlated with agglutinability of these cells with Con A or LcH. Differing ability of Con A and LcH to allow penetration of diaminobenzidine into myeloid cells indicated stearic differences in their binding to the cell surface. The Con A-HRP staining of cell surfaces was prevented on all cells by prior periodate oxidation and on cells other than erythrocytes by prior acetylation but was not altered by methylation or sialidase digestion.

scholarly journals Development of a new monoclonal antibody specific to mouse Vγ6 chain

2019 ◽  
Vol 2 (3) ◽  
pp. e201900363 ◽  
Author(s):  
Shinya Hatano ◽  
Xin Tun ◽  
Naoto Noguchi ◽  
Dan Yue ◽  
Hisakata Yamada ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
Klebsiella Pneumoniae ◽  
Γδ T Cells ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
In Vivo Administration ◽  
Complementarity Determining Region

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene–transfected T-cell line lacking TCR as well as of Vγ1− Vγ4− Vγ5− Vγ7− γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.

Intracellular cytokine detection by flow cytometry in pigs: Fixation, permeabilization and cell surface staining

2007 ◽  
Vol 327 (1-2) ◽  
pp. 18-29 ◽  
Author(s):  
Petra Zelnickova ◽  
Martin Faldyna ◽  
Hana Stepanova ◽  
Jaroslav Ondracek ◽  
Frantisek Kovaru
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Intracellular Cytokine ◽  
Cytokine Detection ◽  
Cell Surface Staining ◽  
Surface Staining
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