Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

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Nanopolymer GluLa-DPG-PEG600-F Can Penetrate into Cells and Deposite in Rats Body

Lesya Ukrainka Eastern European National University Scientific Bulletin Series Biological Sciences ◽

10.29038/2617-4723-2016-337-12-138-142 ◽

2019 ◽

pp. 138-142

Author(s):

Bogdan Chekh ◽

Maria Ferens ◽

Natalia Susol ◽

Sergiy Varvarenko ◽

Dmytro Ostapiv ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Cell Survival ◽

Metabolic Activity ◽

Sperm Cell ◽

Redox Activity ◽

Sperm Cells ◽

Oxygen Intake ◽

Bovine Sperm ◽

Muscle Tissues ◽

High Doses

Via fluorescence microscopy we found that nanopolymer GluLa-DPG-PEG600-F can penetrate bovine sperm cells via binding membranes and deposit in the cite of injection in muscle tissues by binding proteins.Nanopolymer influences on sperm cells metabolic activity decreases oxygen intake and redox activity. We also determined sperm cell survival time, that was decreased of high doses of nanopolymer added to sperm.

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Ca2+ signals obtained with multiple indicators in mammalian vascular muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1987.253.6.h1456 ◽

1987 ◽

Vol 253 (6) ◽

pp. H1456-H1461

Author(s):

T. T. DeFeo ◽

G. M. Briggs ◽

K. G. Morgan

Keyword(s):

Portal Vein ◽

Calcium Concentration ◽

Fluorescent Dyes ◽

Contractile Function ◽

Single Cells ◽

Fluorescent Intensity ◽

Fura 2 ◽

Multiple Indicators ◽

Calcium Storage ◽

Vascular Muscle Cells

Enzymatically isolated single cells from ferret portal vein were loaded with the fluorescent dyes fura-2 and chlortetracycline. Ferret portal vein intact strips were loaded with the luminescent indicator aequorin. At short loading times, fura-2 loading resulted in relatively homogeneous images of labeled cells. At longer loading times, extremely heterogeneous images were obtained that were similar to those produced by chlortetracycline, an indicator recognized to enter calcium-storage organelles. A significant effect of fura-2 on contractile function was detected at the long but not at the short loading time. Caffeine, which is known to deplete calcium from sarcoplasmic reticulum, decreased the fura-2 fluorescent intensity when cells were incubated for a long loading time but caused no statistically significant change at the short loading time. Caffeine caused no drop in the aequorin signal but did cause a drop in the chlortetracycline fluorescence. These results are consistent with the idea that aequorin reports cytoplasmic intracellular calcium concentration ([Ca2+]i), chlortetracycline reports stored calcium, and fura-2 reports a mixed signal from the cytoplasm and calcium-storage organelles depending on incubation time.

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Dimorphisms Observed in Bovine Sperm Cells and Electrophoretic Separation of Types

Fertility and Sterility ◽

10.1016/s0015-0282(16)35413-9 ◽

1964 ◽

Vol 15 (6) ◽

pp. 675-683 ◽

Cited By ~ 1

Author(s):

Cleve W. Laird

Keyword(s):

Sperm Cells ◽

Electrophoretic Separation ◽

Bovine Sperm

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Subcellular quantitative dynamic imaging: from metabolic activity to cell tracking of retinal and corneal structure (Conference Presentation)

Optical Coherence Tomography and Coherence Domain Optical Methods in Biomedicine XXIII ◽

10.1117/12.2511437 ◽

2019 ◽

Author(s):

Jules Scholler ◽

Kassandra Groux ◽

Sacha Reichman ◽

Michel Pâques ◽

Olivier Goureau ◽

...

Keyword(s):

Metabolic Activity ◽

Cell Tracking ◽

Dynamic Imaging ◽

Corneal Structure

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Multicenter performance evaluation of the MultiDrugQuant assay kit.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e21140 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e21140-e21140

Author(s):

Peter Krajcsi ◽

Katalin Tauber Jakab ◽

Sandor Barath ◽

Edit Gyimesi ◽

Zsuzsanna Hevessy ◽

...

Keyword(s):

Multidrug Resistance ◽

Mononuclear Cells ◽

Fluorescent Dyes ◽

Clinical Laboratory ◽

Diagnostic Methods ◽

Cytotoxic Agents ◽

Efflux Transporters ◽

Target Cells ◽

Functional Assay ◽

Fluorescent Intensity

e21140 Background: Multidrug resistance is the most frequent type of resistance to anticancer chemotherapy, which usually results from the overexpression of efflux transporters, such as the MDR1, MRP1 and BCRP. Unfortunatelly, neither the genetic polymorphisms nor the mRNA/protein expression levels correlate closely with the functional activity. On the other hand, although the functional methods separately gave promising results, standardization and reproducibility of these tests failed to conform with values required from routine diagnostic methods. MultiDrugQuant (MDQ) kit was developed as an improved functional assay system, which can measure the multidrug resistance activity of the three, clinically most relevant efflux transporters using flow cytometry in living tumor cells. The present study aimed to carry out the laboratory validation and to evaluate the performance of the MDQ-kit. Methods: Validation of the kit was carried out according to the standards of the Clinical Laboratory Standards Institute in three university centres. Mononuclear cells were separated using Ficoll gradient and tested at 2-5×106/ml within 6 hours after specimen collection. Activities of the multidrug transporters were calculated from the difference between the mean fluorescent intensity of cells w/o the specific inhibitors, respectively. Inaccuracy and comparative measurements were carried out using cell lines with low and high activity of the transporters. Results on different flow cytometers were compared using CD45 CD19 or CD3 monoclonal antibodies for gating the population of interest. Results: The assay proved to be specific and robust at various concentrations of the fluorescent dyes (10-100 % of the original) or inhibitors (50-150 % of the original). Both intraassay and interassay reproducibilities were <5 %. Multidrug resistance activity values determined on different flow cytometers were comparable and eligible. Conclusions: The MDQ assay provides quantitative results on the activity of the MDR1, MRP1 and BCRP in the target cells, which might be used to predict the resistance of these cells to particular cytotoxic agents. Recently, the MDQ-kit has been registered for in vitro diagnostic use in the EU.

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Subcellular Localization of a Novel Alternative Splicing of IIIG9 and Colocalization with PPP1gamma Isoforms

Microscopy and Microanalysis ◽

10.1017/s143192760808968x ◽

2008 ◽

Vol 14 (S3) ◽

pp. 141-143

Author(s):

C. Sousa ◽

A.P. Vintém ◽

M. Fardilha ◽

O. da Cruz e Silva ◽

E. da Cruz e Silva

Keyword(s):

Alternative Splicing ◽

Male Infertility ◽

Cell Types ◽

Sperm Cells ◽

Regulatory Subunits ◽

Specific Antibodies ◽

Fluorescent Microscope ◽

Bovine Sperm ◽

Spermatogonia Cells ◽

Different Cell Types

In testis we find mainly PPP1gamma2 isoform. We hypothesize that in different cell types we can find different regulatory subunits that may constitute targets for therapeutics of diseases such as male infertility, cancer and Alzheimer's disease. We identified a novel alternative splicing isoform of IIIG9 in testis, a known regulator of PPP1, IIIG9sT, and the aim of this study was its further characterization. We used a specific antibody for IIIG9sT in order to characterize its localization in bovine sperm cells. We also transfected IIIG9sT-GFP construct in mouse spermatogonia cells (GC-1 cells) and we used specific antibodies for each PPP1 isoform for the colocalization studies. We observed them under a fluorescent microscope and a LSM and quantified a high co-localization with PPP1gamma1 and 2 isoforms.

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Label-free biochemical characterization of bovine sperm cells using Raman microscopy

10.1117/12.2021575 ◽

2013 ◽

Author(s):

A. C. De Luca ◽

S. Manago ◽

M. A. Ferrara ◽

L. Sirleto ◽

R. Puglisi ◽

...

Keyword(s):

Biochemical Characterization ◽

Raman Microscopy ◽

Sperm Cells ◽

Label Free ◽

Bovine Sperm

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Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3966-3973.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3966-3973 ◽

Cited By ~ 97

Author(s):

Helge M. Ulmer ◽

Michael G. GÃ¯Â¿Â½nzle ◽

Rudi F. Vogel

Keyword(s):

High Pressure ◽

Cell Viability ◽

Membrane Permeability ◽

Lactobacillus Plantarum ◽

Metabolic Activity ◽

Cell Injury ◽

Fluorescent Dyes ◽

Membrane Damage ◽

Food Preservation ◽

Sublethal Injury

ABSTRACT The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 106cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

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