Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

2014 ◽  
Vol 413 ◽  
pp. 45-56 ◽  
Author(s):  
Maren Kuhne ◽  
Martin Dippong ◽  
Sabine Flemig ◽  
Katrin Hoffmann ◽  
Kristin Petsch ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Hybridoma Cells ◽  
Comparative Characterization ◽  
Flow Cytometric

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

1992 ◽  
Vol 8 (1) ◽  
pp. 65-74 ◽  
Author(s):  
José M. Coco-Martin ◽  
Jan W. Oberink ◽  
Tiny A. M. van der Velden-de Groot ◽  
E. Coen Beuvery
Keyword(s):  
Flow Cytometric Analysis ◽  
Suspension Cultures ◽  
Hybridoma Cells ◽  
Flow Cytometric

Characterization of Staphylococcus aureus-Platelet Binding by Quantitative Flow Cytometric Analysis

1992 ◽  
Vol 166 (1) ◽  
pp. 65-73 ◽  
Author(s):  
M. R. Yeaman ◽  
P. M. Sullam ◽  
P. F. Dazin ◽  
D. C. Norman ◽  
A. S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Platelet Binding ◽  
Quantitative Flow

scholarly journals Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts

2019 ◽  
Vol 317 (3) ◽  
pp. H658-H666 ◽  
Author(s):  
Roman Covarrubias ◽  
Mohamed Ameen Ismahil ◽  
Gregg Rokosh ◽  
Tariq Hamid ◽  
Federica Accornero ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Immune Cells ◽  
Cell Separation ◽  
Flow Cytometric Analysis ◽  
Left Ventricular ◽  
Coronary Perfusion ◽  
Flow Cytometric ◽  
Mediators Of Inflammation ◽  
Cell Fixation

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

scholarly journals Transformation of Rickettsia prowazekii to Erythromycin Resistance Encoded by the Escherichia coli ereB Gene

2000 ◽  
Vol 182 (11) ◽  
pp. 3289-3291 ◽  
Author(s):  
Lyudmila I. Rachek ◽  
Andria Hines ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Flow Cytometric Analysis ◽  
Etiologic Agent ◽  
Erythromycin Resistance ◽  
Rickettsia Prowazekii ◽  
Flow Cytometric ◽  
Epidemic Typhus ◽  
Green Fluorescent

ABSTRACT Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression inR. prowazekii of the Escherichia coli ereBgene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene inR. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.

Characterization of exocytosis in electropermeabilized neutrophils by flow cytometric analysis: Difference in sensitivity to calcium and guanosine-5′-[γ-thio]triphosphate

Cytometry ◽  
1994 ◽  
Vol 15 (3) ◽  
pp. 230-236 ◽  
Author(s):  
Ger J. J. C. Boonen ◽  
Ben M. de Koster ◽  
Maarten van der Keur ◽  
John Vansteveninck ◽  
Hans J. Tanke ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Flow Cytometric

scholarly journals Characterization of Thymic Nurse-Cell Lymphocytes, Using an Improved Procedure for Nurse-Cell Isolation

1993 ◽  
Vol 3 (2) ◽  
pp. 103-112 ◽  
Author(s):  
Mireille Lahoud ◽  
David Vremec ◽  
Richard L. Boyd ◽  
Ken Shortman
Keyword(s):  
Nurse Cell ◽  
Flow Cytometric Analysis ◽  
Lymphoid Cells ◽  
Immunomagnetic Bead ◽  
Thymic Nurse Cells ◽  
Flow Cytometric ◽  
Short Period ◽  
High Sedimentation ◽  
Tissue Digestion

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4+8+3lowthymocytes, about 12% of apparently mature CD4+8-3highand CD4-8+3highthymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.

Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis

2014 ◽  
Vol 304 (8) ◽  
pp. 1032-1037 ◽  
Author(s):  
Andreas Wieser ◽  
Enno Storz ◽  
Gabriele Liegl ◽  
Annabell Peter ◽  
Michael Pritsch ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Flow Cytometric Analysis ◽  
Nano Particles ◽  
Flow Cytometric ◽  
Bacterial Outer Membrane

scholarly journals Comparative Analysis of Human Adipose-Derived Mesenchymal Stem Cells from Orbital and Abdominal Fat

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Sarmila Nepali ◽  
Mira Park ◽  
Helen Lew ◽  
Okjoon Kim
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Flow Cytometric Analysis ◽  
Mrna Levels ◽  
Alizarin Red ◽  
Luminex Assay ◽  
Flow Cytometric ◽  
Hla Dr ◽  
Multipotent Mesenchymal Stem Cells

Adipose tissue contains abundant multipotent mesenchymal stem cells with strong proliferative and differentiating potential into adipocytes, osteocytes, and chondrocytes. However, adipose-derived mesenchymal stem cells (ASCs) showed variable characteristics based on the tissue-harvesting site. This study aimed at comparing human adipose-derived mesenchymal stem cell from the orbit (Orbital ASCs) and abdomen (Abdominal ASCs). Orbital and abdominal ASCs were isolated during an upper or lower blepharoplasty operation and liposuction, respectively. Flow cytometric analysis was done to analyze the surface antigens of ASCs, and cytokine profiles were measured using Luminex assay kit. The multilineage potential of both ASCs was investigated using Oil Red O, alizarin red, and alcian staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to measure mRNA levels of genes involved in these trilineage differentiations. Our results showed that both types of ASCs expressed the cell surface markers which are commonly expressed stem cells; however, orbital-ASCs showed higher expressions of CD73, CD90, CD105, and CD146 than abdominal ASCs. Unlikely, orbital-ASC expressed CD31, CD45 and HLA-DR lesser than abdominal-ASCs. Orbital ASCs secreted higher concentrations of eotaxin, fractalkine, IP-10, GRO, MCP-1, IL-6, IL-8, and RANTES but lower MIP-1α, FGF-2, and VEGF concentrations than abdominal-ASCs. Our result showed that orbital ASCs have higher potential towards adipogenic and osteogenic differentiation but lower tendency to chondrogenesis when compared with abdominal ASCs. In conclusion, tissue-harvesting site is a strong determinant for characterization of adipose-derived mesenchymal stem cells. Understanding defining phenotypes of such cells is useful for making suitable choices in different regenerative clinical indications.

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