scholarly journals Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

1977 ◽  
Vol 25 (7) ◽  
pp. 573-579 ◽  
Author(s):  
R H Jensen
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Deoxyribonucleic Acid ◽  
Emission Spectra ◽  
Hoechst 33258 ◽  
Chromomycin A3 ◽  
Fluorescence Excitation ◽  
Specific Staining ◽  
Cervical Cells ◽  
Two Parameter

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.

scholarly journals Sorting and cultivation of Faecalibacterium prausnitzii from fecal samples using flow cytometry in anaerobic conditions

2020 ◽  
Author(s):  
Samuel Bellais ◽  
Mélanie Nehlich ◽  
Aurore Duquenoy ◽  
Maryne Ania ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Polyclonal Antibodies ◽  
Fecal Microbiota ◽  
Anaerobic Conditions ◽  
Specific Staining ◽  
Fecal Samples ◽  
Promising Tool ◽  
Faecalibacterium Prausnitzii ◽  
Efficient Detection

AbstractBackgroundThere is a growing interest in using gut commensal bacteria as ‘next generation’ probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was therefore to adapt flow cytometry and cell sorting to be able to detect, separate, isolate and cultivate new strains of Extremely Oxygen Sensitive (EOS) species from fecal material, focusing on Faecalibacterium prausnitzii as a proof-of-concept.ResultsA BD Influx® cell sorter was equipped with a glovebox that covers the sorting area. This box is flushed with nitrogen to deplete oxygen in the enclosure. Several non-specific staining methods including Wheat Germ Agglutinin (WGA), Vancomycin BODIPY™ and LIVE/DEAD BacLight were evaluated with three different strains of the EOS species F. prausnitzii. In parallel, we generated polyclonal antibodies directed against this species by immunizing rabbits with heat-inactivated bacteria. Anaerobic conditions were maintained during the full process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii reference strains. In addition, staining solutions did not severely impact bacterial viability while allowing discrimination between groups of strains. Efficient detection was achieved using polyclonal antibodies directed against heat-fixed bacteria. Finally, we were able to detect, isolate and cultivate a variety of F. prausnitzii strains from healthy volunteer’s fecal samples using WGA staining and antibodies. These strains present markedly different phenotypes, thus confirming the heterogeneity of the species.ConclusionsCell-sorting in anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations and to sort strains of interest using specific antibodies, thus opening new avenues for targeted culturomics experiments.

scholarly journals Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes.

1977 ◽  
Vol 25 (8) ◽  
pp. 954-964 ◽  
Author(s):  
R H Jensen ◽  
R G Langlois ◽  
B H Mayall
Keyword(s):  
Flow Cytometry ◽  
Energy Transfer ◽  
Ethidium Bromide ◽  
Deoxyribonucleic Acid ◽  
Fluorescent Dyes ◽  
Chinese Hamster ◽  
Fluorescence Properties ◽  
Chromomycin A3 ◽  
Metaphase Chromosomes ◽  
Mitotic Cells

Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.

scholarly journals Flow cytometry of human gynecologic specimens using log chromomycin A3 fluorescence and log 90 degrees light scatter.

1979 ◽  
Vol 27 (1) ◽  
pp. 573-578 ◽  
Author(s):  
D L Barrett ◽  
R H Jensen ◽  
E B King ◽  
P N Dean ◽  
B H Mayall
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Cell Types ◽  
Specific Cell ◽  
Light Scatter ◽  
Chromomycin A3 ◽  
Important Region ◽  
Abnormal Cells ◽  
Cellular Dna ◽  
Microscope Slides

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.

scholarly journals A comparison of mercury arc lamp and laser illumination for flow cytometers.

1979 ◽  
Vol 27 (1) ◽  
pp. 241-245 ◽  
Author(s):  
D C Peters
Keyword(s):  
Light Source ◽  
Fluorescent Dyes ◽  
Emission Spectra ◽  
Light Sources ◽  
Hoechst 33258 ◽  
Chromomycin A3 ◽  
Mouse Sperm ◽  
Laser Illumination ◽  
Spectral Intensities ◽  
Dapi Fluorescence

Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.

Evaluation of p53 protein expression in Barrett's esophagus by two-parameter flow cytometry

1992 ◽  
Vol 102 (4) ◽  
pp. 1220-1228 ◽  
Author(s):  
Stig Ramel ◽  
Brian J. Reid ◽  
Carissa A. Sanchez ◽  
Patricia L. Blount ◽  
Douglas S. Levine ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Barrett’S Esophagus ◽  
Barrett's Esophagus ◽  
P53 Protein ◽  
P53 Protein Expression ◽  
Two Parameter

scholarly journals Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

2002 ◽  
Vol 68 (11) ◽  
pp. 5209-5216 ◽  
Author(s):  
Kaouther Ben Amor ◽  
Pieter Breeuwer ◽  
Patrick Verbaarschot ◽  
Frank M. Rombouts ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Salt Stress ◽  
Bile Salt ◽  
Cell Sorting ◽  
Physiological Activity ◽  
Membrane Integrity ◽  
Multiparameter Flow Cytometry ◽  
Bifidobacterium Adolescentis ◽  
Multiparametric Flow Cytometry ◽  
Plate Counts

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

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