• Korean Chemical Engineering Research
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• 제57권6호
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• pp.898-906
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• 2019

본 연구에서는 전기천공시스템에서 형광 염료를 사용한 세포 생존율 평가와 형광 염료를 전달 물질로 하여 세포 내부로의 전달 효율 평가를 통해 세포의 생존율과 전달 물질의 전달 효율 평가에 사용하기 적합한 형광 염료의 선정 및 사용에 적절한 농도에 대한 가이드라인을 제시 할 수 있는 기초 연구를 수행하였다. 형광 염료로는 Propidium Iodide와 Yo-Pro-1을 사용하였다. Propidium Iodide과 Yo-Pro-1을 사용한 세포 생존율 평가와 전달 효율의 평가에서 각 형광염료의 유세포 분석 히스토그램의 모양은 형광 염료의 종류와 사용한 농도에 따라 다른 것을 확인하였다. 이는 사용하는 형광 염료의 특성과 연관이 있는 것으로 이 결과를 통해 세포 분석에 사용하는 형광 염료의 특성에 따라 분석 결과가 달라지는 것을 알 수 있었다. 형광 염료 자체가 세포 생존률에 미치는 영향은 크지 않음을 확인하였다. 또한 두 종류 이상의 형광 염료를 사용하여 분석을 하는 경우 발생할 수 있는 형광 신호 간의 간섭 영향을 확인하였다. 간섭의 영향은 Yo-Pro-1의 농도가 높을수록 큰 것으로 확인되었으며 Propidium Iodide의 농도는 형광 신호의 간섭에 큰 영향을 미치지 않음을 확인하였다. 본 연구를 통해 형광 염료의 종류와 농도에 따라 세포 생존율 평가와 전달 효율의 평가 결과가 영향을 받는 것을 확인했으며 이는 형광 염료의 특성과 연관이 있는 것으로 판단된다. 또한, 본 연구 결과를 토대로 세포 생존율 평가와 전달 효율 평가에 적절한 형광 염료의 선정과 사용에 적절한 농도를 제시하였다.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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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.

• 한국환경과학회지
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• 제26권1호
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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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• 제14권5호
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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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• 한국생물공학회 2001년도 추계학술발표대회
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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.

• 미생물학회지
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• 제50권4호
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• pp.360-367
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• 2014

Candida albicans는 면역력이 약화된 환자의 표재성에서 전신적 감염까지 다양한 부위에서 감염을 유발시킬 수 있는 기회 감염적 병원성 진균이다. C. albicans는 효모형에서 균사형으로 변환될 수 있으며 이 때 바이오필름을 형성할 수 있다. 바이오필름과 관련된 C. albicans의 감염은 일반적으로 통상적인 항진균제에 대해 내성을 보이므로 새로운 항진균제에 대한 개발이 절실하다. 대황(Rheum undulatum)은 전통적으로 한국과 중국에서 하제나 소염제로 사용되는 약용 식물이다. 본 연구의 목적은 R. undulatum이 캔디다증 환자로부터 분리한 C. albicans 바이오필름 형성 균주에 대한 바이오필름 형성 억제효과와 이에 대한 항진균 활성 기작을 알아보는 것이다. R. undulatum (0.098 mg/ml)은 12종의 바이오필름 형성 임상균주의 캔디다 바이오필름을 $49.4{\pm}6.0%$ 감소시켰고 C. albicans의 폴리스티렌 표면으로의 부착을 억제시켰다. CFDA, AM과 propidium iodide로 이중 염색한 결과 R. undulatum은 C. albicans의 세포막을 손상시켰으며 propidium iodide와 neutral red로 염색하여 공초점 레이저 현미경과 위상차 현미경으로 관찰한 결과 C. albicans의 세포용해를 야기함을 관찰할 수 있었다. Crystal violet 흡수율 실험으로 R. undulatum에 의한 세포막 투과성의 변화를 관찰하였다. 따라서 R. undulatum은 세포막의 손상과 세포막의 투과성 변화로 야기된 세포의 용해와 관련된 항진균 활성이 C. albicans의 바이오필름 형성을 억제하는 것으로 보여진다. 본 연구의 결과는 R. undulatum이 바이오필름과 관련된 캔디다의 감염을 치료하고 제거하기 위한 천연물 기반 항진균제 개발에 대한 좋은 후보물질임을 보여준다.

• 대한한의학회지
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• 제23권3호
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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)

• 대한의생명과학회지
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• 제11권3호
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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.

• 대한한의학방제학회지
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• 제29권4호
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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.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.69-79
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• 2002