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Heliyon Dec 2019This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) tracking of labeled in the fibroblast cells.
BACKGROUND
This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) tracking of labeled in the fibroblast cells.
METHODS
Dil crystal and AO were used to stain in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 10 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 × 10/mL promastigote of was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 μg/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 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 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 in the fibroblast , but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.
PubMed: 31890980
DOI: 10.1016/j.heliyon.2019.e03073 -
Journal of Photochemistry and... Aug 2017Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs,...
Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. Experiments were carried out to examine the susceptibility of C. albicans to PDI. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15J/cm) than in non-irradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by photodynamic inactivation. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains.
Topics: Acridine Orange; Antifungal Agents; Candida albicans; Drug Liberation; Lasers; Liposomes; Microscopy, Atomic Force; Microscopy, Fluorescence; Photosensitizing Agents; Triclosan
PubMed: 28683399
DOI: 10.1016/j.jphotobiol.2017.06.034 -
Methods in Molecular Biology (Clifton,... 2024Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded...
Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded DNA or RNA under ultraviolet light. This unique characterization allows it to be used for distinguishing or visualization of dsRNA. Here, we present a convenient and efficient protocol for detecting dsRNA in polyacrylamide gels.
Topics: Acridine Orange; RNA, Double-Stranded; Staining and Labeling; DNA, Single-Stranded; Ultraviolet Rays
PubMed: 38285384
DOI: 10.1007/978-1-0716-3702-9_2 -
Methods and Protocols Aug 2023Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of...
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.
PubMed: 37623923
DOI: 10.3390/mps6040072 -
Combinatorial Chemistry & High... 2021Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples.
BACKGROUND
Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples.
OBJECTIVE
This study is aimed to make selective, sensitive, simple, fast, and low cost electrochemical analysis of expectorant drug guaifenesin in pharmaceuticals and serum samples.
METHODS
Differential pulse adsorptive stripping voltammetric method for determination of guaifenesin on a poly(acridine orange) modified glassy carbon electrode has been developed. Glassy carbon electrode was modified with electropolymerization of the acridine orange monomer for the sensitive determination of guaifenesin. Guaifenesin provided highly reproducible and welldefined irreversible oxidation peaks at +1.125 V and +1.128 V (vs. Ag/AgCl) in the selected supporting electrolyte and human serum samples, respectively.
RESULTS
Under optimized conditions, linear response of peak current on the concentration of guaifenesin has been obtained in the ranges of 2.00×10-7 to 1.00×10-4 M in Britton Robinson buffer solution at pH 7.0 and 4.00×10-7 to 1.00×10-4 M in serum samples. The precision of the method was detected by intraday and inter-day repeatability studies in the supporting electrolyte and serum samples media.
CONCLUSION
The analytical applicability of the proposed method exhibited satisfying determination results for guaifenesin from pharmaceutical dosage forms (syrup) and human serum samples without any pre-separation procedures.
Topics: Acridine Orange; Carbon; Drug Compounding; Electrochemical Techniques; Electrodes; Guaifenesin; Healthy Volunteers; Humans; Molecular Structure; Pharmaceutical Preparations; Polymers
PubMed: 32646355
DOI: 10.2174/1386207323666200709170450 -
Journal of Orthodontic Science 2022To assess the chemical composition and oral biofilm formation on different types of commercially available clear orthodontic retainer materials (CORM).
AIM
To assess the chemical composition and oral biofilm formation on different types of commercially available clear orthodontic retainer materials (CORM).
MATERIALS AND METHODS
Four types of CORM commercially available were used (Clear advantage series I (CAS1), Clear advantage series II (CAS2), Endure (ES), and CENTRI FORM-clear rigid material (CFCRM)). Circular samples (12 mm diameter) of each CORM were prepared for (n = 40). Unstimulated saliva from twenty volunteers was collected. Fourier Transformation Infrared Spectroscopy (FTIR) was used for the evaluation of the chemical composition of CORM. For the quantitative assessment of oral biofilm formation, samples of each CORM were incubated for twenty-four hours, and crystal violet assay (CVA) was utilized. The degree of absorbance was measured using a spectrophotometer at 570 nm. For qualitative evaluation of oral formation, the samples of each CORM were incubated for 24 hours, and viable biofilm cells stained by acridine orange were examined under a fluorescent microscope.
RESULTS
FTIR findings showed that CAS2 was made of polypropylene and ES is made of polyvinyl chloride, while others were made of co-polyester. CVA results confirmed that CAS2 showed the lowest biofilm formation, which differs significantly compared to CAS1, CFCRM, and ES. No significant difference in biofilm formation was detected between CAS1, CFCRM, and ES. Viable biofilm cells staining by acridine orange showed that CAS2 demonstrated smaller microcolonies of viable biofilm cells compared with CAS1, CFCRM, and ES, which confirmed the result obtained by CVA.
CONCLUSIONS
CAS2 showed anti-microbial activities with a decrease the biofilm formation, which may be related to its chemical composition.
PubMed: 36188210
DOI: 10.4103/jos.jos_7_22 -
Journal of Pharmaceutical Sciences Aug 2022The human papillomavirus (HPV) is responsible for over 90% of all cervical cancer cases. The use of vaginal gels is often indicated for local vaginal drug delivery....
BACKGROUND
The human papillomavirus (HPV) is responsible for over 90% of all cervical cancer cases. The use of vaginal gels is often indicated for local vaginal drug delivery. Previous studies have shown that Thymus vulgaris essential oil (TEO) exhibits anticancer properties besides antifungal and antibacterial properties. Its activity derives from a specific increase in free radicals and oxidative stress caused in cancer cells. Furthermore, mitoxantrone (MTX), an anthracenedione, and C, an acridine orange derivative, were shown to inhibit the growth of the cervical cancer cell line HeLa.
RESULTS
The results showed that TEO + C is the most promising formulation in terms of viscosity and osmolality properties in vaginal fluid simulant (VFS). The combined action of TEO with the compounds MTX and C resulted in HeLa cell viability reduction compared with the effect obtained with the individual formulations containing each one of the compounds.
CONCLUSIONS
The formulation TEO + C holds promise in terms of cost-benefit and topical application of the active compound for the HeLa cells.
Topics: Alphapapillomavirus; Drug Compounding; Female; HeLa Cells; Humans; Oils, Volatile; Papillomaviridae; Uterine Cervical Neoplasms
PubMed: 35182543
DOI: 10.1016/j.xphs.2022.02.004 -
Journal of Oral and Maxillofacial... 2021Oral cancer is a major health problem and its early detection is advantageous for therapeutic purposes. According to available evidence, the risks of oral malignancies...
INTRODUCTION
Oral cancer is a major health problem and its early detection is advantageous for therapeutic purposes. According to available evidence, the risks of oral malignancies increase with the usage of tobacco and other psychoactive substances (PSs). The present study showed expression pattern of nuclear and cytoplasmic changes from normal individuals without habit to oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) in PS abusers with the help of fluorescence acridine orange (AO) stain and Papanicolaou (PAP) stain.
AIM AND OBJECTIVES
This study aimed to investigate and compare diagnostic efficacy of fluorescence microscopic evaluation of AO stain in cytological smears with PAP staining under light microscopy in PS abusers having oral potentially malignant and malignant lesions.
MATERIALS AND METHODS
Oral smears from 120 individuals among which 40 from potentially malignant disorders, 40 from oral malignancy and 40 normal buccal mucosa smears were prepared. One set of smears was stained by AO staining and the other by PAP staining and examined under fluorescence and light microscope, respectively. The results of both the stainings were evaluated by grading cytology smears in class-I to class-V cytology.
RESULTS
The AO fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence. The efficacy of AO fluorescence stain was higher than PAP stain in screening of oral lesions suspicious of malignancy. The sensitivity of PAP staining and AO staining is 57.50% and 61.25%, respectively.
CONCLUSION
As compared to PAP staining method, fluorescent AO method is more effective in screening of OPMD and OSCC in PS abusers.
PubMed: 34349437
DOI: 10.4103/jomfp.JOMFP_275_20 -
Transgenic Research Dec 2021Our study was aimed to investigate the effects of lgals3a (Gal-3 encoding gene) on the development of zebrafish embryo and its underlying mechanisms. Morpholino (MO)...
Our study was aimed to investigate the effects of lgals3a (Gal-3 encoding gene) on the development of zebrafish embryo and its underlying mechanisms. Morpholino (MO) technology was used to inhibit the expression of zebrafish lgals3a, and the effect of lgals3a gene knockdown on zebrafish embryo development and the number of monocyte macrophages was observed. Effect of lgals3a-e3i3-MO on apoptosis of zebrafish was detected by acridine orange staining. In addition, the mRNA expression levels of Wnt/β-catenin signaling pathway-related genes were detected by RT-qPCR. Compared with control-MO group, the zebrafish embryos injected with lgals3a-e3i3-MO had obvious defects in the head, eyes, and tail, and pericardial edema. Lgals3a-e3i3-MO significantly reduced the number of mononuclear macrophages in zebrafish embryos compared with the control-MO group. The results of acridine orange staining showed that compared with the control-MO group, lgals3a-e3i3-MO promoted cardiomyocyte apoptosis in zebrafish. Furthermore, lgals3a-e3i3-MO significantly up-regulated the expression of dkk1b, wnt9a, lrp5, fzd7a, β-catenin, Gsk-3β, mycn, myca in the Wnt/β-catenin pathway, and decreased the expression of lef1. These results indicate that lgals3a-e3i3-MO inhibits zebrafish embryo development, reduces the number of mononuclear macrophages, activates Wnt/β-catenin signaling pathway and promotes cardiomyocyte apoptosis.
Topics: Acridine Orange; Animals; Embryo, Nonmammalian; Embryonic Development; Glycogen Synthase Kinase 3 beta; Receptors, Cell Surface; Wnt Proteins; Wnt Signaling Pathway; Zebrafish; Zebrafish Proteins; beta Catenin
PubMed: 34347236
DOI: 10.1007/s11248-021-00276-5 -
Spectrochimica Acta. Part A, Molecular... Jul 2023Chemotherapy-phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome...
Chemotherapy-phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.
Topics: Daunorubicin; Acridine Orange; Antineoplastic Agents; Drug Delivery Systems; DNA
PubMed: 36905740
DOI: 10.1016/j.saa.2023.122583