• Korean Chemical Engineering Research
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• v.57 no.6
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• pp.898-906
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• 2019

In this study, basic research was conducted to provide guidelines for selecting fluorescent dye and using proper concentration of fluorescent dye to use evaluation of cell viability and fluorescent dye delivery efficiency. Propidium iodide and Yo-Pro-1 were used as fluorescent dyes. In the evaluation of cell viability and the efficiency of delivery using Propidium Iodide and Yo-Pro-1, the histogram of each fluorescent dye was different depending on the type of fluorescent dye and the concentration used. These results were related to the characteristics of the fluorescent dyes used. This was related to the properties of the fluorescent dyes used. From these results, it was found that the analytical results depending on the characteristics of the fluorescent dyes used in the cell analysis. The effect of the fluorescent dye on the cell was confirmed, but it was confirmed that it did not affect the analysis result. In addition, the influence of interference between fluorescent signals was confirmed when two or more kinds of fluorescent dyes were used for analysis. The higher the concentration of Yo-Pro-1 was, the larger the effect of interference was, and the concentration of Propidium Iodide did not affect the interference of fluorescence signal. This study confirmed that the evaluation of the cell viability and the evaluation of the delivery efficiency were influenced by the type and concentration of the fluorescent dyes and it was related to the characteristics of the fluorescent dyes. Based on the results, appropriate concentrations of fluorescent dyes suitable for evaluation of cell viability and delivery efficiency were suggested.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.317-322
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• 2002

Objective: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. Materials and methods: A total 964 blastocysts (early${\sim}$hatched) was exposed to PI (n=831) (group I: $\leq$ 10; II: $11{\sim}15$; III: $16{\sim}20$; IV: $\geq$21 sec) and bisbenzimide (n=133) (group A: $\leq$1; B: $1{\sim}$3; C: $\geq$ 4 hr) in several periods for differential staining. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: In case of PI exposure, differential staining rates were significantly higher (p<0.05) in group I (89.8%) than in any others (group II: 77.6%; III: 29.6%; IV: 22.2%) and higher (p<0.05) in group II than in group III and IV. In case of bisbenzimide exposure, differential staining rates were not statistically differences in three groups (group A: 97.4%; B: 87.8%; C: 93.3%). Conclusion: The differential staining rates of mouse blastocysts are not affected by the exposure length of bisbenzimide. However, blastocysts were exposed to PI with period of shorter than 15 sec show best outcomes of differential staining rates.

• Journal of Environmental Science International
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• v.26 no.1
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• pp.11-21
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• 2017

In this study, bacterial growth was assessed by flow cytometry analysis of fluorescent probes-stained bacteria. Flow cytometry has many advantages of rapid analytical time, a low standard deviation, and highly sensitive detection of live and Dead E.coli over colony forming assay. When untreated bacteria were stained by using Thiazole Orange (TO) and Propidium Iodide (PI), double staining had a short analytical time as compared with that of single staining while its error rate was similar to that of single staining. Through double staining experiments, it was determined that optimal concentrations for TO and PI staining were 420 nM and $9.6{\mu}M$, respectively.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2885-2889
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• 2013

Goniothalamin is an active compound extracted from Goniothalamus griffithii, a local plant found in northern Thailand. Goniothalamin inhibits cancer cell growth but is also toxic to normal cells. The aims of this study were to identify the cytotoxic effect of goniothalamin and the mechanism of cell death in human HL-60 and U937 cells. Cytotoxicity was determined by MTT assay and cell cycle profiles were demonstrated by staining with propidium iodide (PI) and flow cytometry. Apoptosis was confirmed by staining with annexin V-FITC/propidium iodide (PI) and flow cytometry. Reduction of mitochondrial transmembrane potential was determined by staining with dihexyloxacarbocyanine iodide and flow cytometry and expression of Smac, caspase-8 and -9 was demonstrated by Western blotting. Goniothalamin inhibited growth of HL-60 and U937 cell lines. An increase of SubG1 phase was found in their cell cycle profiles, indicating apoptosis as the mode of cell death. Apoptosis was confirmed by the flip-flop of phosphatidylserine using annexin V-FITC/PI assay in HL60 and U937 cells in a dose response manner. Furthermore, reduction of mitochondrial transmembrane potential was found in both cell types while expression of caspase-8, -9 and Smac/Diablo was increased in HL-60 cells. Taken together, our results indicate that goniothalamin-treated human leukemic cells undergo apoptosis via intrinsic and extrinsic pathways.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.918-922
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• 2001

In a previous study, we have isolated a number of lactobacilli from Korean women, and one of them (KLB46) was identified as Lactobacillus crispatus by 16S rRNA gene sequencing. For the ecological treatment of bacterial vaginosis (BV) cell suspension of L. crispatus KLB46 was instillated into BV patients. L. crispatus KLB46 was found to persist for several days in cell suspension with no nutrients. In this study, in order to assess the influence of starvation on physiological activity, we compared the viability and culturability of KLB46 following suspension in various buffer solutions. A pair of in situ fluorescent dye was used to assess viability (i.e. membrane integrity) and the culturability was examined by plate count assay. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit $(BacLight^{TM})$ was applied to estimate both viable and total counts of bacteria in cell suspension. $BacLight^{TM}$ is composed of two nucleic acid-binding stains ($SYTO\;9^{TM}$ and propidium iodide). $SYTO\;9^{TM}$ penetrates all bacterial membranes and stains the cells green while propidium iodide only penetrates cells with damaged membranes, therefore the combination of the two stains produces red fluorescing cells. Optimal staining conditions for $BacLight^{TM}$ were found to be with 0.0835M $SYTO\;9^{TM}$ and 0.05M propidium iodide for 15 min incubation at room temperature in dark. When cells were microscopically examined during 140 hours of starvation, the culturability decreased markedly while the viability remained relatively constant, which suggests that large fraction of KLB46 cells became viable but non-culturable (VBNC) upon starvation.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.360-367
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• 2014

Candida albicans is an opportunistic and the most prevalent fungal pathogen that can cause superficial and systemic infections in immunocompromised patients. C. albicans can promote the transition from budding yeast to filamentous form, generating biofilms. Infections associated with C. albicans biofilms are frequently resistant to conventional antifungal therapy. Therefore, the development of more effective antifungal drugs related with biofilm formation is required urgently. The roots of Rheum undulatum have been used for medicinal purposes in Korea and China traditionally. The aim of present study was to evaluate the effect of R. undulatum extract upon preformed biofilms of 12 clinical C. albicans isolates and the antifungal activities. Its effect on preformed biofilms was evaluated using XTT reduction assay, and metabolic activity of all tested strains was reduced significantly ($49.4{\pm}6.0%$) at 0.098 mg/ml R. undulatum. The R. undulatum extract blocked the adhesion of C. albicans biofilms to polystyrene surfaces, and damaged the cell membrane integrity of C. albicans which was analyzed by CFDA, AM, and propidium iodide double staining. It caused cell lysis which was observed by Confocal laser scanning and phase contrast microscope after propidium iodide and neutral red staining, respectively. Membrane permeability was changed as evidenced by crystal violet uptake. The data suggest that R. undulatum inhibits biofilm formation by C. albicans, which can be associated with the damage of the cell membrane integrity, the changes in the membrane permeability and the cell lysis of C. albicans.

• The Journal of Korean Medicine
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• v.23 no.3
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• pp.145-163
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• 2002

Objectives: The purpose of this investigation was to evaluate the effects of Woohwangcheongsim-won and to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20 (E20) cortical cells of a rat (Sprague Dawley). Methods: E20 cortical cells were dissociated in neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Woohwangcheongsim-won was added to the culture media for 24 hrs or 72 hrs. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Woohwangcheongsim-won's effects for neuronal death protection were evaluated by LDH assay, propidium iodide stain and phospho-H2AX immunostain and the mechanisms were studied by Bcl-2, Bak, Bax, caspase family, PKCα, ca1pain I. Results & Conclusions : 1. This study indicated that Woohwangcheongsim-won's effects for neuronal death protection in hypoxia were confirmed by LDH assay, propidium iodide stain and phospho-H2AX immunostain in culture method of Embryonic day 20(E20) cortical neuroblasts. 2. Woohwangcheongsim-won's mechanisms for neuronal death protection in hypoxia are to reduce the membrane damage fraction, to restrain DNA truncate, to restrain inflow of cytochrome c into cellularity caused by Bak diminution, to reduce the caspase cascade intiator caspase-8 and the effector caspase-3, to reduce the calpain I activity and to increase PKCand its activity in the membrane fraction. (J Korean Oriental Moo 2002;23(3):145～163)

• Biomedical Science Letters
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• v.11 no.3
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• pp.281-287
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• 2005

Although actinomycin D (AMD) is known to induce apoptotic cell death to various cell lines, the mechanism of apoptosis induced by AMD is still unclear. Understanding this mechanism may improve its therapeutic efficacy. The present study has been performed to elucidate expression of Bcl-2 and Caspase-3 proteins related to apoptosis in human leukemia K-562 cells. Five different assays were performed in this study; DNA fragmentation analysis by agarose gel electrophoresis, quantitative assay of fragmented DNA, morphological assessment of apoptotic cells, quantification of apoptosis by annexin V (AV) and propidium iodide (PI) staning, and expression of Bcl-2 and Caspase-3 proteins by the western blot analysis. The number of apoptotic cells and amount of fragmented DNA in this cell line treated with AMD was increased at 6 hour. DNA ladder pattern was also appeared at 6 hour. The expression of Bcl-2 was decreased, and disappeared from 12 hours after AMD treatment. Precursor of Caspase-3 was degraded, and 20 kDa cleavage products were detected. These results suggest that AMD induced apoptosis of K-562 cells is Caspase-3-dependent fashion, and this apoptosis is related to the degradation of Bcl-2 proteins.

• Herbal Formula Science
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• v.29 no.4
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• pp.267-275
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• 2021

Objectives : Previously, we reported that compound K isolated from fermented ginseng by Aspillus oryzae has a wide biochemical and pharmacological effect, including anti-cancer activity in prostate cancer PC-3 cells. Despite these findings, its signaling pathway and gene expression pattern are not clearly understood. Methods : To confirm the gene expression study of treated with compound K in PC-3 cells, a cDNA microarray chip composed of 44K human cDNA probes was used. MTT assay, western blot analysis, propidium iodide staining, and annexin V/propidium iodide staining were analyzed. Results : We confirmed the differences of gene expression profiles. Then, we analyzed with the cell cycle arrest, cell death and cell proliferation related genes using DAVID database. Conclusions : Our finding should be useful for understanding genome-wide expression patterns of compound K-mediated cell cycle arrest toward induction of cell death and be helpful for finding future cancer therapeutic targets for prostate cancer cells.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2002.11a
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• pp.69-79
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• 2002