Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula

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.

Download Full-text

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

Journal of Immunological Methods ◽

10.1016/j.jim.2014.07.004 ◽

2014 ◽

Vol 413 ◽

pp. 45-56 ◽

Cited By ~ 9

Author(s):

Maren Kuhne ◽

Martin Dippong ◽

Sabine Flemig ◽

Katrin Hoffmann ◽

Kristin Petsch ◽

...

Keyword(s):

Flow Cytometric Analysis ◽

Hybridoma Cells ◽

Comparative Characterization ◽

Flow Cytometric

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.182.11.3289-3291.2000 ◽

2000 ◽

Vol 182 (11) ◽

pp. 3289-3291 ◽

Cited By ~ 37

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.

Download Full-text

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

Cytotechnology ◽

10.1007/bf02540031 ◽

1992 ◽

Vol 8 (1) ◽

pp. 65-74 ◽

Cited By ~ 9

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

Download Full-text

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

Cytometry ◽

10.1002/cyto.990150308 ◽

1994 ◽

Vol 15 (3) ◽

pp. 230-236 ◽

Cited By ~ 6

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

Download Full-text

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

European Food Research and Technology ◽

10.1007/s00217-021-03881-0 ◽

2021 ◽

Author(s):

Lorenza Dalla Costa ◽

Daniela Vinciguerra ◽

Lisa Giacomelli ◽

Umberto Salvagnin ◽

Stefano Piazza ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Gene Transfer ◽

Molecular Characterization ◽

Good Practice ◽

Integrated Approach ◽

Digital Pcr ◽

Plant Genome ◽

Practical Case ◽

Knock Out

AbstractAgrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

Download Full-text

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

Developmental Immunology ◽

10.1155/1993/43482 ◽

1993 ◽

Vol 3 (2) ◽

pp. 103-112 ◽

Cited By ~ 6

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.

Download Full-text