• BMB Reports
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• 제29권2호
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• pp.127-132
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• 1996

Germinal centers (GCs) are formed in peripheral lymphoid tissues in response to protein antigens. In order to see if immunoglobulin isotype switching takes place in GC B-cells, we isolated GC B-cells (PNA positive cells) from mouse popliteal lymph nodes by a flow cytometer after the staining of lymph node cells with PNA-FITC and anti-B220-PE, and determined the expression of ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA by RT-PCR. ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA were amplified specifically in cDNAs from hybridoma expressing IgG1 or splenocytes stimulated LPS plus IL-4. Germinal center B-cells formed in popliteal lymph nodes of mice immunized with chicken ovalbumin were isolated 7 days after immunization. We sorted GC B-cells five times. Immunoglobulin ${\gamma}1$ germline transcripts were expressed in germinal center B-cells in three out of five sorts whereas two out of five sorts did not express ${\gamma}1$ germline transcripts in GC B-cells. The contents of GC B-cells ranged from 5 to 7% of total lymph node cells in most flow cytometric analyses but those of two sorted cells which did not express ${\gamma}1$ germline transcripts were out of normal range. These results imply that isotype switching of immunoglobulins may take place in GCs.

• 동의생리병리학회지
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• 제26권4호
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• pp.441-445
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• 2012

Cadmium is an important occupational and environmental pollutant that damages various organs, especially renal proximal tubular cells. We examined the effect of aqueous extract of Buddleja officinalis (ABO) on cadmium chloride ($CdCl_2$)-induced cytotoxicity in HK-2 human renal proximal tubular cells. HK-2 cells were preincubated with ABO (50, 100, 200 and 400 ${\mu}g/ml$) for 3 hr and then treated with 10 ${\mu}M$ $CdCl_2$ for 24 hr. The effect of ABO on $CdCl_2$-induced cytotoxicity in HK-2 cells was investigated by using MTT assay, morphological observation, flow cytometric analysis and Western blot. The results of the MTT assay and morphological observation indicated that $CdCl_2$-induced cytotoxicity was prevented by pretreatment with ABO. In flow cytometric analysis, ABO reduced sub-G1 peak (apoptotic peak) in $CdCl_2$-treated cells. $CdCl_2$-induced procaspase-3 proteolysis and PARP cleavage reduced by pretreatment with ABO. These results suggest that ABO effectively inhibited $CdCl_2$-induced cytotoxicity in HK-2 cells.

• Journal of Acupuncture Research
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• 제17권2호
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• 대한한의학회지
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• 제20권1호
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• pp.91-105
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• 1999

The effects of Yinjin and Yinjinsaryongsangagambang on a DNA damaging agent, etoposide-induced apoptosis, cell viability, cell cycle progression, and mRNA expression of apoptosis-related genes of human hepatocyte cell line HepG2 were investigated using tryphan blue exclusion assay, MTT assay, flow cytometry, immunocytometric analysis of PCNA, and quantitative RT-PCR analysis. MTT assay showed that Yinjin and Yinjinsaryongsangagambang increases cellular viability of HepG2 cells in a dosage-dependent manner. Stimulation of cell cycle progression by Yinjin or Yinjinsaryongsangagambang was detected by flow cytometric analysis of the DNA content and immunocytometric analysis of PCNA expression. A significant reduction of a DNA-damaging agent, etoposide-induced apoptosis were found in both Yinjin and Yinjinsaryongsangagambang-treated cells in dosage-dependent manner. In overall, 3-fold reduction of apoptosis was recognized in $10.0\;{\mu}g/ml$ of Yinjin or Yinjinsaryongsangagambang-treated cells compared to untreated cells. Although the difference is not significant, Yinjinsaryongsangagambang showed slightly higher effect on the inhibition of apoptosis than Yinjin. From flow cytometric analysis of apoptosis, while 39.9% of untreated cells showed etoposide-induced apoptotic cell death, only 19.6% or 17.4% of Yinjin or Yinjinsaryongsangagambang-treated cells were fond at apoptotic sub G1 phase, respectively. Interestingly, strong induction of Gadd45-mRNA was observed from Yinjin or Yinjinsaryongsangagambang-treated cells. However, no changes in expression levels of p53 and Waf1 were detected, demonstrating that induction of Gadd45 mRNA expression by Yinjin or Yinjinsaryongsangagambang occurs by p53-independent mechanism. Marked mRNA inductions of two apoptosis-inhibiting genes, Bcl-2 and Bcl- XL, were found in both Yinjin or Yinjinsaryongsangagambang-treated HepG2 cells while no changes was detected in expression levels of an apoptosis-promoting gene, Bax.

• 전기학회논문지
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• 제56권12호
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• pp.2208-2213
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• 2007

In this study we have developed a hyperspectrum imaging system for highly sensitive and effective imaging analysis. An optical setup was designed using acoustic optical tunable filter (AOTF) for high sensitive hyperspectrum imaging. Light emitted by mercury lamp gets split in to diffracted and undiffracted beams while passing though AOTF. GFP transfected HEK-293 cell line was used as a model for in vitro imaging analysis. Cells were first, analyzed by fluorescence microscope followed by flow cytometric analysis. Flow cytometric analysis showed 66.31% transfection yield in GFP transfected HEK-293 cells. Various images of GFP transfected HEK-293 cell were grabbed by collecting the diffracted light using a CCD over a dynamic range of frequency of 129-171 MHz with an interval of 3 MHz. Subsequently, for in vivo image analysis of GFP transfected cells in mouse, a whole-body-imaging system was constructed. The blue light of 488 nm wavelength was obtained from a Xenon arc lamp using an appropriate filter and transmitted through an optical cable to a ring illuminator. To check the efficacy of the newly developed whole-body-imaging system, a comparative imaging analysis was performed on a normal mouse in presence and absence of Xenon arc irradiation. The developed hyperspectrum imaging analysis with AOTF showed the highest intensity of green fluorescent protein at 153 MHz of frequency and 494 nm of wavelength. However, the fluorescence intensity remained same as that of the background below 138 MHz (475 nm) and above 162 MHz (532 nm). The mouse images captured using the constructed whole-body-imaging system appeared monochromatic in absence of Xenon arc irradiation and blue when irradiated with Xenon arc lamp. Nevertheless, in either case mouse images appeared clearly.

• 대한소아치과학회지
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• 제23권2호
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• pp.503-524
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• 1996

A series of 31 patients with primary oral squamous cell carcinoma (SCC) who were treated at Chonbuk National University Hospital during the years 1991-1995, were evaluated by dual parameter analysis in flow cytometric DNA measurement, Bryne's malignancy grading system, and the TNM classification. The aims of the present study were to discover that previously undetected aneuploid clones could be detected by dual parameter analysis and to determine the prognostic value of the above parameters. 1. Using dual parameter analysis of cytokeratin and DNA on disintegrated paraffin-embedded samples, aneuploid clones which were undetected by regular single parameter DNA analysis could be found among the cytokeratin-selected cells. DNA aneuploidy from paraffin-embedded samples were 15 cases compared with 10 cases using conventional DNA analysis. 2. The portion of aneuploid tumors showed slightly higher clinical stage and tumor size than the portion of diploid tumors, but the difference was not significant. The portion of DNA aneuploid tumors showed significantly higher mean mitosis and total malignancy scores than the portion of DNA diploid tumors. 3. The majority of the patients presented with clinical stage III and IV lesions showed significantly higher mean total malignancy score as compared to those with clinical stage I and II. 4. Histopathologic mean total malignancy score of the 31 cases was 12.7. Among the histologic parameters, mean mitosis score was correlated to the status of DNA ploidy and total malignancy score were correlated to the DNA ploidy and clinical staging.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.151-154
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• 2000

배큘로바이러스와 actinomycin D에 의해 유도되는 곤충세포 apoptosis의 누에체액에 의한 저해 효과에 대해 연구하였다. 감염 전 또는 감염 중, 배양 배지에 누에체액을 첨가함으로써 배큘로바이러스에 의해 감염된 숙주 세포의 생존율을 높은 수준으로 유지시켜 주었다. 누에체액은 또한, RNA 합성 저해제인 actinomycin D에 의해 유도된 apoptosis 역시 저해시켰다. 이러한 효과들은 TUNEL assay와 flow cytometry로 확인하였다. 추가적으로, 누에체액은 감염 후 세포 내 유전자의 발현에는 영향을 미치지 않으나 바이러스 유전자의 발현을 증진시킴을 알 수 있었다.

• International Journal of Oral Biology
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• 제30권2호
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• pp.65-69
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• 2005

Periodontitis is a multi-microbial disease and the comparison of a series of periodontopathogenic and non-periodontopathogenic bacteria in terms of microbe-host interaction may provide clues to understand the microbial etiology of the disease better. When we deal with twenty different bacterial species in a study, the first technical issue is how to measure the accurate concentration and use the same number of bacterial cells. We measured bacterial concentration by enumerating bacteria stained with SYTOX green for constant time using a flow cytometer and compared the results with those obtained by plate counting. Concentrations calculated by two different methods were very close. Therefore, flow cytometric counting allowed the rapid analysis of live/dead bacteria, offering the advantage of turbidity measurement and that of colony counting together.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.607-612
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• 2003

To elucidate the action mechanism of MCS-C2, a novel analogue of toyocamycin and sangivamycin, its effect on the expression of cell cycle-related proteins in the human myelocytic leukemia cell line HL-60 was examined using Western blotting and a flow cytometric analysis. MCS-C2, a selective inhibitor of cyclin-dependent kinases, was found to inhibit cell growth in a time- and dose-dependent manner, and inhibits cell cycle progression by inducing the arrest at G1 and G2/M phases, in HL-60 cells. The flow cytometric analysis revealed an appreciable arrest of cells in the G2/M phase of the cell cycle after treatment with MCS-C2. The HL-60 cell population increased gradually from 13% at 0 h, to 28% at 12 h in the G2/M phase, after exposure to $2{\;}\mu\textrm{M}$ MCS-C2. Furthermore, Western blot analysis demonstrated that MCS-C2 induced the cell cycle arrest at G1 phase through the inhibition of pRb phosphorylation. Hypophosphorylated pRb accumulated after treatment with $5{\;}\mu\textrm{M}$ MCS-C2 for 12 h, whereas, the level of hyperphosphorylated pRb was reduced. Thus, treatment of the cell with MCS-C2 suppressed the hyperphosphorylated form of pRb with a commensurate increase in the hypophosphorylated form.

• 생명과학회지
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• 제20권2호
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• pp.253-259
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• 2010

본 연구는 땅콩나물 추출물이 glutamate가 유도하는 세포독성으로부터 신경세포를 보호하는 효과를 확인하였다. 땅콩나물을 부위별로 전체, 머리, 줄기 부분으로 나누어 methanol로 처리하여 얻어진 각각의 추출물 WME, HME 그리고 SME를 이용하여 glutamate에 의하여 유도된 세포독성에 대한 신경세포보호효과를 관찰하였다. 기지의 신경세포 N18-RE-105 세포주를 이용하여 MTT reduction assay, LDH release assay, 형태학적인 변화 및 apoptosis를 관찰한 결과로부터 HME에서 효율적인 신경세포보호효과를 보였다. 다음으로 HME를 이용하여 hexane, diethyl ether, ethyl acetate, water 층으로 분획하여 신경세포 보호 효과를 확인한 결과, diethyl ether 층에서 가장 높은 활성을 확인할 수 있었다. 그리고 HME의 apoptosis 억제 효과를 확인하기 위하여 flow cytometric analysis를 실시한 결과에서 glutamate 만을 처리하였을 경우 sub-G1기 세포가 58.5%의 확인되었으나 HME를 100 mg/ml 동시 처리하였을 때에는 sub-G1 세포가 9.1%로 감소하여 높은 apoptosis 억제 효과를 확인할 수 있었다. 이상의 결과로부터 땅콩나물 머리 부분 methanol 추출물에는 glutamate에 의한 세포독성으로부터 신경세포를 보호하는 효과가 있다는 것을 알 수 있었다.