• 제목/요약/키워드: Flow cytometric analysis

검색결과 295건 처리시간 0.034초

저산소성 허혈성 뇌손상이 유발된 신생백서에서 단일세포의 분리 (Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain)

  • 황종희;성동경;최창원;강샘;장윤실;박원순;이문향
    • Clinical and Experimental Pediatrics
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    • 제48권5호
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    • pp.545-550
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    • 2005
  • 목 적: 저산소성 허혈성 손상을 받은 신생아의 뇌조직에서 유세포 방법을 통해 세포의 사멸을 분석하기 위해서는 단일세포의 분리가 이루어져야 한다. 본 연구는 세포 분리에 있어서 세포막의 소실을 최소화하고 항원성을 유지하기 위하여 물리적인 방법과 효소 처리를 통한 세포 분리방법의 효율성에 대해 알아보고자 하였다. 방 법 : 생후 7일된 10마리의 SD 신생백서에 우측 경동맥 결찰 후, 8% 산소에 노출시켜 저산소성 허혈증의 손상을 유발하였으며 48시간이 지난 후 뇌조직을 얻어 같은 수의 정상 대조군과 비교하였다. 세포 분리는 물리적인 방법(pipette)과 효소 처리(trypsin 및 collagenase) 방법을 통하여 이루어 졌으며, 세포막의 손상 정도와 범위에 대해서는 annexin V 및 propidium iodide의 형광 염색을 통한 유세포 분석방법을 이용하였다. 결 과 : 정상 대조군에서, 물리적인 방법을 통한 세포 분리가 양반구 모두에서 효소 처리를 한 경우에 비해서 세포의 사멸과 괴사가 통계적으로 유의하게 증가하였다. 저산소성 허혈증을 유발한 군 중, collagenase를 이용하여 세포 분리를 시행한 경우에서 우측 반구의 세포 사멸과 괴사의 비율이 좌측 반구 및 정상 대조군 보다 유의하게 증가하였다. 효소 처리를 통한 세포 분리에서는 서로 유사한 경향을 보였으나, trypsin을 이용한 경우가 collagenase를 이용한 경우에 비해 세포 변화의 정도가 유의하게 감소하였다. 결 론 : 신생아의 뇌조직에서 collagenase를 이용한 단일 세포 분리방법이 세포막의 손상을 최소화하면서 세포막의 성상을 보존할 수 있는 가장 유용한 방법이었다.

Characterization of a Novel Gene in the Extended MHC Region of Mouse, NG29/Cd320, a Homolog of the Human CD320

  • Park, Hyo-Jin;Kim, Ji-Yeon;Jung, Kyung-In;Kim, Tae-Jin
    • IMMUNE NETWORK
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    • 제9권4호
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    • pp.138-146
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    • 2009
  • Background: The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17. Methods: We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis. Results: The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line. Conclusion: These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.

Induction of Apoptosis by Gagamhwanglyeonhaedog-tang through Activation of Caspase-3 in Human Leukemia Cell Line HL-60 Cells

  • Park Sang Goo;Won Jin Hee;Kim Dong Woung;Moon Goo
    • 동의생리병리학회지
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    • 제18권4호
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    • pp.1173-1178
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    • 2004
  • Gagamhwanglyeonhaedog-tang(GHH), a Korean genuine medicine, is a newly designed herbal drug formula based on the traditional oriental pharmacological knowledge for the purpose of treating tumorous diseases. Apoptosis is an evolutionarily conserved suicide program residing in cells. It leads to cell death through a tightly regulated process resulting in the removal of damaged or unwanted tissue. In the present study, the apoptosis inducing activities of the decocted water extract of GHH were studied. Results of the 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay showed that GHH had a strong cytotoxic effect on HL-60 cells. The number of live cells was less than 20% after exposure to 1㎎/㎖ GHH for 48 hr. GHH increased cytotoxicity of HL-60 cells in a dose- and time­dependent manner. Cell apoptosis by GHH was confirmed by flow cytometric analysis of the DNA-stained cells. The percentage of apoptotic cells increased to 28%, 31% and 37% 24 hr and 37%, 44% and 81% 48 hr after treatment with 0.01, 0.1 and 1㎎/㎖ GHH, respectively. Flow cytometric analysis of GHH treated HL-60 cells showed increase of hypodiploid apoptotic cells in a dose- and time- dependent manner. DNA fragmentation also occurred in apoptosis and was characterized by a ladder pattern on agarose gel. In addition, GHH (0.01 and 0.1㎎/㎖) increased the secretion of tumor necrosis factor-alpha in 24 and 48 hr. The author showed that GHH-induced apoptosis was accompanied by activation of caspase-3. These results suggest that GHH induces activation of caspase-3 and eventually leads to apoptosis.

Cadmium Induces Cell Cycle Arrest and Change in Expression of Cell Cycle Related Proteins in Breast Cancer Cell Lines

  • Lee Young Joo;Kang Tae Seok;Kim Tae Sung;Moon Hyun Ju;Kang Il Hyun;Oh Ji Young;Kwon Hoonjeong;Han Soon Young
    • Toxicological Research
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    • 제21권1호
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    • pp.77-85
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    • 2005
  • Cadmium is an environmental pollutant exposed from contaminated foods or cigarette smoking and known to cause oxidative damage in organs. We investigated the cadmium-induced apoptosis and cell arrest in human breast cancer cells, MCF-7 cells and MDA-MB-231 cells. Obvious apoptotic cell death was shown in CdCl₂ 100 μM treatment for 12 hr, which were determined by DAPI staining and flow cytometric analysis. In cell cycle analysis, MCF-7 cells and MDA-MB-231 cells were arrested in S phase and G2/M phase respectively. These could be explained by the induction of cell cycle inhibitory protein, p21/sup Waf1/Cip1/ and p27/sup Kip1/, expression and reduction of cyclin/Cdk complexes in both cell lines. The decreased expression of cyclin A and Cdk2 in MCF-7 cells and cyclin B1 and Cdc2 in MDA-MB-231 cells were consistent with the flow cytometric observation. p-ERK expression was increased dose-dependent manner in both cell lines. It suggests that ERK MAPK pathway are involved in cadmium-induced cell cycle arrest and apoptosis. Moreover, cotreatment of zinc (100 μM, 12 hr) recovered the cadmium-induced cell arrest in both cells, which shows cadmium-induced oxidative stress mediates apoptosis and cell cycle arrest in human breast cancer cells.

수 종의 한약제제가 조혈작용에 미치는 영향 (Effects of Korean Traditional Medicine on Murine Hematopiesis (Regulation of Hematopoietic Cytokine & $CD34^{+}$ cell Expression))

  • 전재현;김영철;이장훈;우흥정
    • 대한한의학회지
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    • 제22권3호
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    • pp.156-168
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    • 2001
  • Objectives : To evaluate the diverse actions of stimulation on the hematopoietic system, 4 formulas (KH I, KH 2, KH 3, KH 4) were studied. Method and Result : RT-PCR was performed to measure the gene expression of hematopoietic cytokines (TPO, GM-CSF, SCF, IL-3). When bone marrow cells were treated with KH 1, 2, 3, 4, the gene expressions of TPO, SCF, IL-3, and GM-CSF were increased. Flow cytometric analysis was performed to measure the expression of CD34+ cell activity. After 72 hrs culture supplemented with KH 1, 2, 3, 4, the percent of CD34+ cell of KH 2, 3, 4 were increased. To measure the expression of colony forming units - granulocyte erythrocytes, macrophages, megakaryocytes (CFU-GEMM) and burst forming unit-erythroid (BFU-E), semisolid clonogenic assay was performed. After 14 days of culture the number of CFU-GEMM and BFU-E of KH I, 2, 3, 4 were significantly increased compared to those of EPO groups (KH 1 P<0.0l, KH 2 P<0.05, KH 3 P<0.001, KH 4 P<0.0l). To determine the intracelluar TPO expression by KH 3, KH 4 in bone marrow cells, intracelluar staining and flow cytometric analysis were performed. After 24 hrs cultures, the TPO expression of the KH 3 and KH 4 treated groups were increased over those of the controlled groups (control : 50%, KH 3 : 87%, KH 4 : 78%). Conclusion : These results suggest that KH I, KH 2, KH 3, KH 4 have hematopoietic effects through increasing the production of hematopoietic cytokines and stimulating the activity of $CD34^{+}$ cells. This study also shows that KH 3 has a more effective hematopoietic effect than KH 1, 2, 4. These results suggest that the formulas (KH I, 2, 3, 4) can be applied to the patients with inappropriate hematopoietic system, and that KH 3 can be the most effective formula among these 4 in treating bone marrow disease in clinics.

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인간 백혈병 세포에서 Psuedomonas aeruginosa exotoxin A에 대한 세포독성과 세포자멸사 효과 (The Cytotoxic and Apoptotic Effect of Pseudomonas aeruginosa Exotoxin A on Human Leukemia K-562 Cells)

  • Chang, Jeong-Hyun;Kwon, Heun-Young
    • 대한임상검사과학회지
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    • 제39권2호
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    • pp.68-75
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    • 2007
  • 약 100년 전에 박테리아가 암을 억제한다는 보고를 바탕으로 다양한 미생물이 항암효과를 가지는 백신 개발에 이용되거나 또는 미생물의 세포 밖 독소 단백질을 찾아내고 있다. Psuedomonas aeruginosa exotoxin A(ETA)는 암세포에서 세포성장을 억제하고 세포 죽음을 유발하는 것으로 알려져 있다. 하지만 ETA가 세포 자멸사를 유도하는 정확한 기전은 아직 알려져 있지 않다. 따라서 본 연구에서는 세포자멸사의 유도를 확인하기 위해 K562 cell을 이용하여 세포의 형태학적 변화, 세포독성, Annexin-V binding assay 그리고 세포주기를 분석하였으며, 그 결과로 ETA는 K-562세포에서의 세포증식과 성장을 억제하였고, 세포자멸사 기작을 통한 K-562 암세포의 사멸을 일으켰음을 관찰하였다. 또한 flow cytometric analysis에서는 ETA가 세포주기 중 특히 sub-G1 기를 정지시키는 것으로 나타났다. 본 연구는 ETA가 인간 백혈병 K-562 암세포의 세포성장을 억제하고 sub-G1 기를 정지시킴으로서 세포자멸사를 유도하고 있음을 확인하였다.

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Flow Cytometer를 이용한 소 과립막세포의 분석 : 난포성숙에 따른 세포주기의 변화 (Flow Cytometric Analysis of Bovine Granulosa Cells : Changes of Cell Cycle During Follicular Maturation)

  • 김해정;김동훈;이훈택;정길생
    • 한국가축번식학회지
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    • 제17권4호
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    • pp.279-285
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    • 1994
  • The objective of the present study was to characterize the cell cycles of granulosa cell populations during follicular maturation in cattle by using flow cytometer. Granulosa cells were isolated from bovine preovulatory antral follicles of F1(>10mm), F2(5~20mm), F3(3~4mm) and F4(1~2mm) diameter and fixed and stained with fluorochromes that selectively bine to DNA. Flow cytometer equipped with a laser excitation system was used to analyze the intensity of fluorescence from stained cells. Forward angle light-scatter(FSC) and 90$^{\circ}$light-scatter(SSC) signals were adopted to measure the size and the granularity of granulosa cells. As a results of FSC/SSC analysis, granulosa cell populations(G1 phase of cell cycle) from each follicle were relatively regular in size and granularity, regardless of follicular size. However, their distribution in granularity was greater than that in size. Most of granulosa cell populations collected from each follicle were distributed in G0/G1, S and G2/M phases. As the follicles approached to ovulation the percentage of cells in the proliferative phases of cell cycle (S and G2/M) decreased significantly, but there was a concomitant increase in the percentage of granulosa cells in G1 phase. Therefore, these data indicate the proportion of main populations to cell cycle of granulosa cells may be changed from proliferative phase to G1 phase during follicular maturation in cattle.

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Characterization of a Novel Monoclonal Antibody (27H2) Recognizing Human CD34 Class III Epitope

  • Hong, Kwon-Pyo;Kang, Sung-Hee;Lee, Kyoung-Mee;Ji, Gil-Yong;Yoon, Sang-Soon;Kim, Jong-Suk;Son, Bo-Ra;Lee, Dong-Geun;Lee, Ok-Jun;Song, Hyung-Geun
    • IMMUNE NETWORK
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    • 제10권6호
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    • pp.239-246
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    • 2010
  • Background: Monoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia. Methods: 27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done. Results: Only KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity. Conclusion: A novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

Anticancer Effects of Curcuma C20-Dialdehyde against Colon and Cervical Cancer Cell Lines

  • Chaithongyot, Supattra;Asgar, Ali;Senawong, Gulsiri;Yowapuy, Anongnat;Lattmann, Eric;Sattayasai, Nison;Senawong, Thanaset
    • Asian Pacific Journal of Cancer Prevention
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    • 제16권15호
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    • pp.6513-6519
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    • 2015
  • Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of $65.4{\pm}1.74{\mu}g/ml$, $58.4{\pm}5.20{\mu}g/ml$ and $72.0{\pm}0.03{\mu}g/ml$, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

기내배양에서 도깨비고비와 참쇠고비의 전엽체 형태형성 (Prothallus Morphogenesis of Cyrtomium falcatum (L.) Presl and Cyrtomium caryotideum var. coreanum Nakai In vitro Culture)

  • 정진아;이철희
    • 한국자원식물학회지
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    • 제19권2호
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    • pp.360-364
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    • 2006
  • 도깨비고비와 참쇠고비의 포자발아에서 얻은 전엽체를 균질화하여 배양함으로써 전엽체 개체발생과 포자체 형성과정을 연구하였다. 배양 2주일이 지나자 균질화한 전엽체 세포들이 일차원의 필라멘트 형태를 형성하였고, 4주일째에는 다수의 가지를 뻗은 형태의 전엽체가 출현하였다. 배양 6주일째에 apical notch가 발달된 전엽체가 형성되었는데. 중심부에는 분열조직이 발달되었다. 8주의 배양기간이 지나자 전엽체의 중심부에서 장란기의 형성 없이 무수정생식에 의한 bud가 관찰되었다. 10주일이 지나자 무수정생식에 의한 bud는 포자체로 발달되었는데, flow cytometric 분석 결과 도깨비고비와 참쇠고비 모두에서 전엽체와 포자체는 동일한 ploidy level을 지닌 것으로 확인되었다. 이는 도깨비고비뿐만 아니라 참쇠고비 또한 무수정생식을 하는 양치식물임을 증명하는 결과로 생각된다.