• Title/Summary/Keyword: flow cytometry

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Bcl-2 Overexpression Inhibits Generation of Intracellular Reactive Oxygen Species and Blocks Adriamycin-induced Apoptosis in Bladder Cancer Cells

  • Kong, Chui-Ze;Zhang, Zhe
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.2
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    • pp.895-901
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    • 2013
  • Resistance to induction of apoptosis is a major obstacle for bladder cancer treatment. Bcl-2 is thought to be involved in anti-apoptotic signaling. In this study, we investigated the effect of Bcl-2 overexpression on apoptotic resistance and intracellular reactive oxygen species (ROS) generation in bladder cancer cells. A stable Bcl-2 overexpression cell line, BIU87-Bcl-2, was constructed from human bladder cancer cell line BIU87 by transfecting recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. The sensitivity of transfected cells to adriamycin (ADR) was assessed by MTT assay. Apoptosis was examined by flow cytometry and acridine orange fluorescence staining. Intracellular ROS was determined using flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were also investigated by the xanthinoxidase and visible radiation methods using SOD and CAT detection kits. The susceptibility of BIU87-Bcl-2 cells to ADR treatment was significantly decreased as compared with control BIU87 cells. Enhanced expression of Bcl-2 inhibited intracellular ROS generation following ADR treatment. Moreover, the suppression of SOD and CAT activity induced by ADR treatment was blocked in the BIU87-Bcl-2 case but not in their parental cells. The overexpression of Bcl-2 renders human bladder cancer cells resistant to ADR-induced apoptosis and ROS might act as an important secondary messenger in this process.

Associations Between Age, Cytogenetics, FLT3-ITD, and Marrow Leukemia Cells Identified by Flow Cytometry

  • Su, Long;Gao, Su-Jun;Tan, Ye-Hui;Han, Wei;Li, Wei
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.9
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    • pp.5341-5344
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    • 2013
  • Objectives: To explore the relationships between age, cytogenetic subgroups, molecular markers, and cells with leukemic aberrant immunophenotype in patients with acute myeloid leukemia (AML). Methods: In this study, we evaluated the correlations between age, cytogenetic subgroups (normal, balanced and unbalance karyotype), molecular mutations (NPM1, FLT3-ITD, and CEBPA mutations) and marrow leukemia cells (LC) identified by flow cytometry in 256 patients with de novo AML. Results: From age group 10-19 years to age group ${\geq}60$ years, the percentage of LC decreased from $67.0{\pm}18.4%$ to $49.0{\pm}25.1%$ (F=2.353, P=0.041). LC percentage was higher in patients with balanced karyotypes ($65.7{\pm}22.4%$), than those with unbalanced karyotypes ($46.0{\pm}26.6%$) (u=3.444, P=0.001) or a normal karyotype ($49.9{\pm}22.1%$) (u=5.093, P<0.001). Patients with FLT3-ITD ($64.3{\pm}19.5%$) had higher LC percentages compared with those without ($54.2{\pm}24.3%$) (u=2.794, P=0.007). Conclusions: Associations between age, cytogenetics, molecular markers, and marrow leukemia cells may offer beneficial information to understand the biology and pathogenesis of AML.

Bacterial Surface Display of $GFP_{UV}$ on Bacillus subtilis Spores

  • Kim, Jung-Hyung;Roh, Chang-Hyun;Lee, Chang-Won;Kyung, Do-Hyun;Choi, Soo-Keun;Jung, Heung-Chae;Pan, Jae-Gu;Kim, Byung-Gee
    • Journal of Microbiology and Biotechnology
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    • v.17 no.4
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    • pp.677-680
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    • 2007
  • To analyze a cotG-based Bacillus subtilis spore display system directly, $GFP_{UV}$ was expressed on the surface of Bacillus subtilis spores. When $GFP_{UV}$ was fused to the C-terminal of the cotG structural gene and expressed, the existence of a $CotG-GFP_{UV}$ fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This $GFP_{UV}$ displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.

Orostachys japonicus DW and EtOH Extracts Induce Apoptosis in Cholangiocarcinoma Cell Line SNU-1079

  • Choi, Eun Sol;Lee, Jang Hoon
    • The Journal of Korean Medicine
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    • v.36 no.4
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    • pp.19-34
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    • 2015
  • Objectives: This study was performed to investigate the anti-tumor effect of O. japonicus extracts on intrahepatic cholangiocarcinoma cell line SNU-1079. Methods: Cholangiocarcinoma SNU-1079 cells were treated with various concentrations of O. japonicus DW and EtOH extracts ($0-300{\mu}g/ml$) for 24, 48 or 72 h. Cell viability was evaluated through a PMS/MTS assay, and the apoptosis rate was examined through ELISA assay and flow cytometry analysis. The mRNA expression of apoptosis- and cell cycle progression-related genes (Bcl-2, Mcl-1, Bax, Survivin, Cyclin D1, and p21) was evaluated using real-time PCR, and the caspase activity was examined using immunoblot analysis. Results: O. japonicus extracts inhibited cell proliferation and increased apoptosis rate in both ELISA assay and flow cytometry analysis. O. japonicus extracts decreased Bcl-2, Mcl-1, Survivin, and Cyclin D1 mRNA expression and increased Bax mRNA level. O. japonicus extracts also increased Caspase-3 activation. Overall, O. japonicus DW extracts were more effective than EtOH extracts. Conclusions: O. japonicus inhibited cell proliferation and induced apoptosis in SNU-1079 cells via mitochondria -mediated intrinsic pathway, which leads to Caspase-3 activation. The results indicate that O. japonicus is a potential therapeutic herb with anti-tumor effect against intrahepatic cholangiocarcinoma.

Expression of Bacillus macerans Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae.

  • Kim, Gyu-Yong;Kim, Myeong-Dong;Han, Nam-Su;Seo, Jin-Ho
    • 한국생물공학회:학술대회논문집
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    • 2000.11a
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    • pp.191-193
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    • 2000
  • A whole-cell biocatalyst was constructed by immobilizing an enzyme on the surface of the yeast Saccharomyces cerevisiae. The gene encoding Bacillus macerans cyclodextrin glucanotransferase(CGTase) was fused with the AGA2 gene encoding a small peptide disulfide-linked to the aga1, a cell wall protein of a-agglutinin. The plasmid was introduced S. cerevisiae and expressed in the medium consisting of 10g/L yeast extract, 20g/L peptone, and 20g/L galactose. The activity was detected with the formation of cyclodextrin(CD) from 10g/L soluble starch. Surface display of CGTase was also verified with the halo-test, flow cytometry, and immunofluorescence microscopy. The recombinant S. cerevisiae produced ${\alpha}-cyclodextrin$ more efficiently than the free CGTase by simultaneous fermentation and cyclization as yeast consumes glucose and maltose which are inhibitors for CD synthesis.

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CD3+/CD4+/CD5+/CD8+/CD21+/CD34-/CD45-/CD79a-/TCRαβ+/TCRγδ-/MHCII+ T-zone lymphoma in a dog with generalized lymphadenopathy: a case report

  • Shin, Sun Woo;Lim, Yu jin;Bae, Hyeona;Kim, Jihu;Cho, ARom;Park, Jinho;Lee, Dongbin;Jung, Dong-In;Kim, Sang-ki;Yu, DoHyeon
    • Korean Journal of Veterinary Research
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    • v.61 no.3
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    • pp.21.1-21.6
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    • 2021
  • Canine T-zone lymphoma (TZL) is a mature T-cell lymphoma in dogs. The diagnosis and sub-classification are impossible without biopsy or immunophenotyping by flow cytometry. An 11-year-old, spayed, female Golden Retriever presented with lymph node enlargement. Clinical examination was consistent with canine multicentric lymphoma. However, immunophenotyping revealed positive for CD3, CD4, CD5, CD8, CD21, TCRαβ, and MHCII but negative for CD34, CD45, CD79a, and TCRγδ. Histopathology revealed lymphocytes expanding to the cortex-preserving architecture and thinning of the nodal capsule, and CD3 positive but PAX-5 negative. Owing to the indolent nature of TZL, careful monitoring approach without clinical intervention was utilized.

Perspective on Rapid and Selective Method for Detecting Microbiology in Dairy Industry: A Review (낙농산업에 필요한 미생물 검사방법과 전망: 총설)

  • Chon, Jung-Whan;Kim, Hyun-Sook;Kim, Hong-Seok;Kim, Dong-Hyeon;Song, Kwang-Young;Yim, Jin-Hyuk;Choi, Dasom;Lim, Jong-Soo;Jeong, Dong-Gwan;Kim, Soo-Ki;Seo, Kun-Ho
    • Journal of Dairy Science and Biotechnology
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    • v.33 no.2
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    • pp.119-127
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    • 2015
  • To date, detection of microbial populations in dairy products has been performed using culture media, which is a time-consuming and laborious method. The recently developed chromogenic media could be more rapid and specific than classical culture media. However, the newly developed molecular-based technology can detect microbial populations with greater rapidity and sensitivity than the classical method involving culture media and chromogenic media. This molecular-based technology could provide various options for monitoring the characterization of different states of bacteria and cells. Thus, it could help upgrade the processing system of the dairy industry so as to maintain the safety and quality of dairy foods. Among the various newly developed molecular-based technologies, flow cytometry can potentially be used for monitoring microbiological populations in the dairy industry if official international standards are available for this purpose. When omics technology would have biomarker identification, it could be regarded as the rapid and sensitive analytical methods. Methods based on PCR, which has become a basic technique in microbiological research, can be developed and validated as alternative methods for quantification of dairy microorganisms. This review discusses methods for monitoring microbiological populations in dairy foods and the limitations of these studies, as well as the need for further research on such methods in the dairy industry.

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Improvement of Chromosome Doubling Efficiency in Cymbidium Hybrids by Colchicine and Oryzalin Treatment (Colchicine과 oryzalin 처리에 의한 Cymbidium hybrids의 기내 배수화 효율 증진)

  • Hwang, Sook-Hyun;Kim, Mi-Seon;Park, So-Young
    • Horticultural Science & Technology
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    • v.33 no.6
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    • pp.900-910
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    • 2015
  • This study aimed to investigate the efficiency of colchicine and oryzalin in inducing polyploidy in two Cymbidium hybrids [Showgirl 'Silky' and Mystery Island 'Silk Road' (Silk Road-4)]. Colchicine was used at concentrations ranging from 50 to $500mg{\cdot}L^{-1}$, with treatments lasting 1 to 3 weeks. Oryzalin was used at concentrations ranging from 3 to $20mg{\cdot}L^{-1}$, with treatments lasting 3 to 6 days or 1 to 3 weeks. The survival rate of PLBs was better in colchicine than in oryzalin solutions. The ploidy levels were screened using flow cytometry. In C. Showgirl 'Silky', the highest chromosome doubling efficiencies were obtained with the 1-week treatment in $50mg{\cdot}L^{-1}$ colchicine (60%) and the 2-week treatment in $5mg{\cdot}L^{-1}$ oryzalin (46.7%). In C. Mystery Island 'Silk Road' (Silk Road-4), the highest chromosome doubling efficiencies were obtained with the 1-week treatment in $50mg{\cdot}L^{-1}$ colchicine (16.7%) and the 3-day treatment in $10mg{\cdot}L^{-1}$ oryzalin (6.7%). Colchicine was more efficient than oryzalin in terms of polyploidy induction. Furthermore, pre-treatment, which entailed poking 10 times with forceps, improved the efficiency of chromosome doubling.

Comparison of Physiological Characteristics, Stomata and DNA Content between Seedling and 5-year-old Somatic Plant (Somatic Embryo Derived-plant) in Liriodendron tulipifera (백합나무 5년생 실생묘 및 체세포묘 (체세포배 유래 식물체) 간의 생리적 요인, 기공 및 DNA 함량 비교)

  • Kim, Yong Wook;Moon, Heung Kyu
    • Journal of Korean Society of Forest Science
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    • v.102 no.4
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    • pp.537-542
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    • 2013
  • Field performance of somatic plants (somatic embryo derived-plants) of yellow-poplar (Liriodendron tulipifera) produced from somatic embryogenesis was compared with that of seedlings at age 5. In comparison of photosynthetic rate (seedling, $10.67{\mu}mol$ $CO_2m^{-2}s^{-1}$; somatic plant, $9.04{\mu}mol$ $CO_2m^{-2}s^{-1}$), stomatal conductance rate (seedling, 0.2 $H_2Om^{-2}s^{-1}$; somatic plant, 0.166 $H_2Om^{-2}s^{-1}$) and respiration rate (seedling, 1.71 mmol $H_2Om^{-2}s^{-1}$; somatic plant, 1.513 mmol $H_2Om^{-2}s^{-1}$), no significant differences were found between plants. The seedlings were a little higher in comparison of stomatal density (seedling, $23.33/mm^2$; somatic plant, $22.43/mm^2$), length (seedling, $25.83{\mu}m$; somatic plant, $23.46{\mu}m$) and width (seedling, $15.87{\mu}m$; somatic plant, $15.3{\mu}m$). In comparison of DNA content of the leaves using flow cytometry, no differences in ploidy level were found between the seedlings and somatic plants.

Effect of Sheath Fluid with HEPES on Viability of Sex-sorted Sperm in Hanwoo (Korean Native Cattle) (한우 정자와 성 분리 시 HEPES를 첨가한 Sheath Fluid가 생존율에 미치는 영향)

  • Lee, Ji-Eun;Lee, Kyung-Jin;Yoo, Han-Jun;Park, Joung-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.26 no.3
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    • pp.181-186
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    • 2011
  • Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.