Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM). The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

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Toxic and osmotic effects of glycerol on human granulocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1984.247.5.c382 ◽

1984 ◽

Vol 247 (5) ◽

pp. C382-C389 ◽

Cited By ~ 28

Author(s):

W. J. Armitage ◽

P. Mazur

Keyword(s):

Plasma Membrane ◽

Osmotic Stress ◽

Cell Volume ◽

Membrane Integrity ◽

Fluorescein Diacetate ◽

Major Fraction ◽

Plasma Membrane Integrity ◽

Human Granulocytes ◽

Osmotic Effects

Human granulocytes are damaged by exposure to concentrations of glycerol as low as 0.5 M. We therefore investigated the addition of glycerol to granulocytes and its subsequent dilution under various conditions to try to distinguish between toxic and harmful osmotic effects of glycerol. The lesion caused by glycerol at 0 degree C was expressed as a loss of plasma membrane integrity (as visualized by fluorescein diacetate) only after incubation (greater than or equal to 1 h) at 37 degrees C. This damage was not ameliorated when osmotic stress was lessened by reducing the rates of addition and dilution of glycerol to keep the computed cell volume within 80-170% of isotonic cell volume. However, when osmotic stress was reduced further by increasing the temperature of addition and dilution of glycerol from 0 degree C to 22 degrees C, the tolerance of the cells to 1 M glycerol increased somewhat. Reducing exposure to glycerol to 3 min or less at 0 degree C greatly increased survival, but this time was too short to allow glycerol to equilibrate intracellularly. Finally, the presence of extra impermeant solute (NaCl or sucrose) in the medium to reduce the equilibrium cell volume to 60% of isotonic cell volume enabled granulocytes to survive 30-min exposure to 1 M glycerol at 0 degree C, but cells had to remain shrunken during the 37 degrees C incubation to prevent the loss of membrane integrity. Suspensions that contained damaged granulocytes formed aggregates when incubated at 37 degrees C, and these aggregates were responsible for a major fraction of the observed loss in viability.

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

Applied and Environmental Microbiology ◽

10.1128/aem.68.11.5209-5216.2002 ◽

2002 ◽

Vol 68 (11) ◽

pp. 5209-5216 ◽

Cited By ~ 204

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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Use of fluorescent staining and flow cytometry for monitoring physiological changes in solventogenic clostridia

Anaerobe ◽

10.1016/j.anaerobe.2013.10.006 ◽

2014 ◽

Vol 29 ◽

pp. 113-117 ◽

Cited By ~ 8

Author(s):

Petra Patakova ◽

Michaela Linhova ◽

Pavla Vykydalova ◽

Barbora Branska ◽

Mojmir Rychtera ◽

...

Keyword(s):

Flow Cytometry ◽

Physiological Changes ◽

Fluorescent Staining ◽

Solventogenic Clostridia

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The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Fitopatologia Brasileira ◽

10.1590/s0100-41582006000400004 ◽

2006 ◽

Vol 31 (4) ◽

pp. 349-356 ◽

Cited By ~ 5

Author(s):

Luiz G. Chitarra ◽

Peter Breeuwer ◽

Tjakko Abee ◽

Ruud W. Bulk

Keyword(s):

Flow Cytometry ◽

Fluorescent Probes ◽

Membrane Integrity ◽

Pathogenic Bacterium ◽

Plant Pathogenic Bacterium ◽

Clavibacter Michiganensis ◽

Clavibacter Michiganensis Subsp ◽

Alternatives Assessment ◽

Heat Treated ◽

Calcein Am

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

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Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽

10.2217/nnm-2020-0056 ◽

2020 ◽

Vol 15 (20) ◽

pp. 1965-1980

Author(s):

Teresa Vilanova-Perez ◽

Celine Jones ◽

Stefan Balint ◽

Rebecca Dragovic ◽

Michael L Dustin ◽

...

Keyword(s):

Flow Cytometry ◽

Mitochondrial Membrane ◽

Membrane Integrity ◽

Computer Assisted ◽

Sperm Function ◽

Sperm Analysis ◽

Mammalian Sperm ◽

Boar Sperm ◽

Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

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