Spectroscopic investigation of increased fluorescent intensity of fluorescent dyes when adsorbed onto polystyrene microparticles

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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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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Optimization of PTFE Coating on PDMS Surfaces for Inhibition of Hydrophobic Molecule Absorption for Increased Optical Detection Sensitivity

Sensors ◽

10.3390/s21051754 ◽

2021 ◽

Vol 21 (5) ◽

pp. 1754

Author(s):

Junyi Yao ◽

Yiyang Guan ◽

Yunhwan Park ◽

Yoon E Choi ◽

Hyun Soo Kim ◽

...

Keyword(s):

Fluorescent Dyes ◽

Lipid Production ◽

Optical Detection ◽

Nile Red ◽

Porous Matrix ◽

Detection Sensitivity ◽

Microfluidic Chips ◽

Fluorescent Intensity ◽

Lipid Signal ◽

Significant Difference

Polydimethylsiloxane (PDMS) is a polymer widely used for fabrication and prototyping of microfluidic chips. The porous matrix structure of PDMS allows small hydrophobic molecules including some fluorescent dyes to be readily absorbed to PDMS and results in high fluorescent background signals, thereby significantly decreasing the optical detection sensitivity. This makes it challenging to accurately detect the fluorescent signals from samples using PDMS devices. Here, we have utilized polytetrafluoroethylene (PTFE) to inhibit absorption of hydrophobic small molecules on PDMS. Nile red was used to analyze the effectiveness of the inhibition and the absorbed fluorescence intensities for 3% and 6% PTFE coating (7.7 ± 1.0 and 6.6 ± 0.2) was twofold lower compared to 1% and 2% PTFE coating results (17.2 ± 0.5 and 15.4 ± 0.5). When compared to the control (55.3 ± 1.6), it was sevenfold lower in background fluorescent intensity. Furthermore, we validated the optimized PTFE coating condition using a PDMS bioreactor capable of locally stimulating cells during culture to quantitatively analyze the lipid production using Chlamydomonas reinhardtii CC-125. Three percent PTFE coating was selected as the optimal concentration as there was no significant difference between 3% and 6% PTFE coating. Intracellular lipid contents of the cells were successfully stained with Nile Red inside the bioreactor and 3% PTFE coating successfully minimized the background fluorescence noise, allowing strong optical lipid signal to be detected within the PDMS bioreactor comparable to that of off-chip, less than 1% difference.

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Spectroscopic investigation of polymers dyed with fluorescent dyes ? Naphthalimide derivatives

Fibre Chemistry ◽

10.1007/bf00551625 ◽

1994 ◽

Vol 25 (5) ◽

pp. 356-359

Author(s):

Z. D. Tul'guk ◽

R. N. Nurmukhametov ◽

V. G. Khimenko ◽

L. I. Semenova

Keyword(s):

Spectroscopic Investigation ◽

Fluorescent Dyes

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.4.1900872 ◽

1991 ◽

Vol 39 (4) ◽

pp. 485-489 ◽

Cited By ~ 25

Author(s):

T W Downing ◽

D L Garner ◽

S A Ericsson ◽

D Redelman

Keyword(s):

Metabolic Activity ◽

Fluorescent Probes ◽

Cell Tracking ◽

Fluorescent Dyes ◽

Oxygen Metabolism ◽

Sperm Cells ◽

Fluorescent Intensity ◽

Cell Staining ◽

Bovine Sperm ◽

Fluorescent Stains

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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Some Results of the Spectroscopic Study of Four Close Binary Systems of Early Spectral Types

International Astronomical Union Colloquium ◽

10.1017/s025292110002755x ◽

1965 ◽

Vol 5 ◽

pp. 120-130

Author(s):

T. S. Galkina

Keyword(s):

Spectroscopic Study ◽

Spectral Type ◽

Spectroscopic Investigation ◽

Binary Systems ◽

Quantitative Information ◽

Close Binary ◽

Physical Parameters ◽

Absorption And Emission ◽

Close Binary Systems ◽

Quantitative Estimates

It is necessary to have quantitative estimates of the intensity of lines (both absorption and emission) to obtain the physical parameters of the atmosphere of components.Some years ago at the Crimean observatory we began the spectroscopic investigation of close binary systems of the early spectral type with components WR, Of, O, B to try and obtain more quantitative information from the study of the spectra of the components.

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Conformation of Ribosomes from Escherichia Coli

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051062 ◽

1974 ◽

Vol 32 ◽

pp. 210-211

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Fluorescent Dyes ◽

Protein Biosynthesis ◽

Three Dimensional ◽

Spatial Arrangement ◽

Dimensional Structure ◽

Complete Understanding ◽

And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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Analysis of 3-d molecular distribution with the digital imaging microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102286 ◽

1988 ◽

Vol 46 ◽

pp. 40-41

Author(s):

W.A. Carrington ◽

F.S. Fay ◽

K.E. Fogarty ◽

L. Lifshitz

Keyword(s):

Image Restoration ◽

Digital Imaging ◽

Fluorescent Dyes ◽

Optical Axis ◽

Computer Hardware ◽

Computer Algorithms ◽

Low Contrast ◽

Specific Proteins ◽

Effective Use ◽

Single Living Cells

Advances in digital imaging microscopy and in the synthesis of fluorescent dyes allow the determination of 3D distribution of specific proteins, ions, GNA or DNA in single living cells. Effective use of this technology requires a combination of optical and computer hardware and software for image restoration, feature extraction and computer graphics.The digital imaging microscope consists of a conventional epifluorescence microscope with computer controlled focus, excitation and emission wavelength and duration of excitation. Images are recorded with a cooled (-80°C) CCD. 3D images are obtained as a series of optical sections at .25 - .5 μm intervals.A conventional microscope has substantial blurring along its optical axis. Out of focus contributions to a single optical section cause low contrast and flare; details are poorly resolved along the optical axis. We have developed new computer algorithms for reversing these distortions. These image restoration techniques and scanning confocal microscopes yield significantly better images; the results from the two are comparable.

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High-resolution measurement of birefringent fine structure in living cells using a new polarized light microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136647 ◽

1995 ◽

Vol 53 ◽

pp. 54-55

Author(s):

Rudolf Oldenbourg

Keyword(s):

Light Microscope ◽

Fluorescent Dyes ◽

Polarized Light ◽

Processing System ◽

Video Camera ◽

Molecular Organization ◽

Computer Assisted ◽

Image Recording ◽

Birefringent Crystal ◽

Technological Advances

The recent renaissance of the light microsope is fueled in part by technological advances in components on the periphery of the microscope, such as the laser as illumination source, electronic image recording (video), computer assisted image analysis and the biochemistry of fluorescent dyes for labeling specimens. After great progress in these peripheral parts, it seems timely to examine the optics itself and ask how progress in the periphery facilitates the use of new optical components and of new optical designs inside the microscope. Some results of this fruitful reflection are presented in this symposium.We have considered the polarized light microscope, and developed a design that replaces the traditional compensator, typically a birefringent crystal plate, with a precision universal compensator made of two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at ALL POINTS of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen.

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En face dual fluorescence staining of fatty streaks allows observation of initiation and progression of atherosclerotic lesions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140695 ◽

1995 ◽

Vol 53 ◽

pp. 864-865

Author(s):

Anne M. Klinkner ◽

Crystal R. Waites ◽

Peter J. Bugelski ◽

William D. Kerns

Keyword(s):

Fluorescent Dyes ◽

Nile Red ◽

Dimethyl Formamide ◽

High Cholesterol Diet ◽

Methods Development ◽

Atherosclerotic Lesions ◽

En Face ◽

Biochemical Evaluation ◽

Stock Solutions ◽

Dual Staining

A primary effort in the understanding of the progression of atherosclerotic disease has been methods development for visualization of the atherosclerotic plaque. We introduce a new method for the qualitative analysis of lipids in atherosclerotic fatty streaks which also retains those lipids for biochemical evaluation. An original aspect of the process is the ability to view an entire fatty streak en face, selectively stained for specific lipid classes within the lesion.New Zealand white rabbits were fed a high cholesterol diet(0.15%-0.3% for 14 wks). The aorta was removed and fixed in Carson's phosphate buffered formaldehyde followed by dual staining in the fluorescent dyes Nile red and filipin. Stock solutions of nile red(0.5mg/ml acetone) and filipin(2.5mg/ml dimethyl formamide) were prepared and kept at -20°C; all subsequent steps were at RT. 0.5cm × 1.0cm pieces of aorta were trimmed and adventitia removed. The pieces were then washed 3×15 min in PBS w/o CaMg, soaked in Nile red(NR)/filipin(Fl) stain(100(il NR stock + 200μl Fl stock in 10 ml PBS for 30 min, washed in PBS 3×30 min, rinsed with distilled water, mounted(Crystal Mount, Biomedia) and coverslipped and viewed by fluorescence microscopy.

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