Analysis of glycosaminoglycans in urine by using acridine orange fluorescence.
Ontology highlight
ABSTRACT:
SUBMITTER: Diakun GP
PROVIDER: S-EPMC1186105 | biostudies-other | 1978 Nov
REPOSITORIES: biostudies-other
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Project description:Hepatic proteolysis is inhibited by cell swelling following a variety of experimental manoeuvres, such as reduction of extracellular osmolarity, concentrative uptake of amino acids, or blockade of K+ channels by barium. On the other hand, proteolysis is known to be accomplished by pH-sensitive lysosomal proteases. Accordingly, NH3/NH4+ inhibits proteolysis by intralysosomal alkalinization. The present study has been performed to test for an effect of cell volume on the pH of acidic intracellular compartments, as assessed by Acridine Orange fluorescence at > 520 nm (F > 520). F > 520 is enhanced by NH3/NH4+ (2 and 20 mmol/l respectively), by glutamine (2 mmol/l), by the K(+)-channel blocker barium (10 mmol/l) and by reduction of extracellular osmolarity (by 20 and 80 mosmol/l respectively). The observations point to release of Acridine Orange from acidic cellular compartments, which is indicative of alkalinization of these compartments during cell swelling. This effect may contribute to the regulation of proteolysis.
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Project description:Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.
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Project description:Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.
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Project description:Simultaneous non-invasive visualization of blood vessels and nerves in patients can be obtained in the eye. The retinal vasculature is a target of many retinopathies. Inflammation, readily manifest by leukocyte adhesion to the endothelial lining, is a key pathophysiological mechanism of many retinopathies, making it a valuable and ubiquitous target for disease research. Leukocyte fluorography has been extensively used in the past twenty years; however, fluorescent markers, visualization techniques, and recording methods have differed between studies. The lack of detailed protocol papers regarding leukocyte fluorography, coupled with lack of uniformity between studies, has led to a paucity of standards for leukocyte transit (velocity, adherence, extravasation) in the retina. Here, we give a detailed description of a convenient method using acridine orange (AO) and a commercially available scanning laser ophthalmoscope (SLO, HRA-OCT Spectralis) to view leukocyte behavior in the mouse retina. Normal mice are compared to mice with acute and chronic inflammation. This method can be readily adopted in many research labs.
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Project description:Background:This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) in vitro tracking of labeled L. major in the fibroblast cells. Methods:Dil crystal and AO were used to stain L. major in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 106 parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The stained promastigote was incubated with fibroblasts for 6-8 h. The presence of stained parasites with AO in the fibroblast was assessed using a fluorescence microscope. 1 × 106/mL promastigote of L. major was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 ?g/mL and it was kept for 20 min at the room temperature. Subsequently, after washing it in PBS for seven times, it was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the precipitate containing stained promastigote was suspended in fresh DMEM F12 with fibroblasts at 37 °C for 6 h. The presence of stained parasites with Dil in fibroblast was assessed using a fluorescence microscope. Fibroblast characterization was undertaken by a real-time polymerase chain reaction (PCR). Results:Acridine orange staining assisted in detection of the live parasite in the fibroblast cells. Free promastigote looked green before entering into the fibroblasts after 12 h culture. The parasite entered the cytoplasm of fibroblasts at the beginning of the exposure and gradually entered the nucleus of the fibroblast. The fibroblast nucleus was entirely stained green by AO. The L. major appeared green under the fluorescent microscope. Dil staining revealed that the internalized parasites with red/orange color were localized within the cytoplasm after 6-hours and the nucleus of the fibroblasts after 72-hours following culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative for CD106 and integrin alpha 11. Conclusion:The fluorescent dye Dil staining is a safe, easy to use, inexpensive and fast method for labeling of the Leishmania parasite in the fibroblast cells. Acridine orange staining could be useful for tracing the parasites in the fibroblasts too. In this study, both Dil and AO were compared and considered as suitable vital dyes for identifying labeled Leishmania in the fibroblast in vitro, but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.
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Project description:BACKGROUND:Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. METHODS:The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. RESULTS:Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. CONCLUSIONS:Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.
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Project description:Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 μM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.
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