Flow Cytometry에 의한 개 신선정액과 동결정액의 생존성 분석

홍유미,김용준,유일정,지동범,김명순 한국동물생명공학회(구 한국동물번식학회) 2004 Reproductive & developmental biology Vol.28 No.3

Flow cytometry를 이용하여 개 정자의 생존율 평가를 수행하고자 2-4세의 수캐 5두가 이용되었고, 분석을 위해 PI염색을 실시하였다. Flow cytometry를 이용한 개 신선 정액의 생존율 평가는 생존 정자와 죽은 정자의 비율을 1:0, 1:1, 1:3으로 조성하여 이를 flow cytometry로 평가하고 광학현미경검사, CFDA/PI 염색검사, HOS test에 의한 생존율과 비교하여 flow cytometry와의 상관관계를 알아보았다. 또한 개 정액을 동결하여 응해 후의 개 정자의 생존율 평가에도 동일한 방법으로 상관관계를 조사하였다. 신선 정액에서 생존 정자와 죽은 정자의 비율이 1:0, 1:1, 1:3 모든 경우에서 flow cytometry를 이용한 생존율은 HOS test에 의한 생존율과 높은 상관관계를 나타내었다 (p<0.01). 신선 정액에서 생존 정자와 죽은 정자의 비율이 1:0과 1:3일 때 광학현미경적 검사에 의한 생존율은 flow cytometry 분석에 의한 생존율과 유의 적인 상관관계를 나타내었으나 (p<0.05), 1:1 비율의 경우 상관관계를 보이지 않았다. 신선 정액에 생존 정자와 죽은 정자의 비율이 1:0과 1:1일 때 CFDA/PI 염색 검사에 의한 생존율은 flow cytometry분석에 의한 생존율과 높은 상관관계를 보였으며(p<0.01), 1:3 비율에서는 유의적인 상관관계를 보였다 (p<0.05). 동결 및 응해 후의 개 정자의 생존율 평가에서 HOS test 결과는 flow cytometry분석에 의한 생존율과 높은 상관관계를 보였으며 (p<0.01), 광학현미경적 검사를 통한 생존율은 유의적인 상관관계를 보였으나 (p<0.05), CFDA/PI 염색 검사결과는 상관관계를 보이지 않았다. 이상의 결과 flow cytometry는 신선정액 및 동결ㆍ융해 후 개 정자에 대한 생존율 검사에 정확한 평가 방법 인 것으로 판단되었다. This study was performed to examine the correlations among dog sperm viabilities evaluated by flow cytometry, by microscopic evaluation (ME), by carbo-xifluorescein diacetate and propidium iodide (CFDA/PI) and by hypoosmotic swelling (HOS) test. Semen were collected from 5 dogs ranging in age from 2 to 4 years. Each ejaculate was divided into 3 aliquots and different proportions of freeze-killed cells were added to each aliquot (1:0, 1:1 and 1:3). In the other experiment, semen was extended with Sweden extender containing 5% glycerol and equex STM paste, and frozen using liquid nitrogen vapor. Fresh and frozen-thawed dog sperm viability were assessed by flow cytometry using PI staining method. The accuracy of flow cytometry was evaluated by comparing with other classic assessments, microscopic evaluation, epifluorescence microscopic analysis using CFDA/PI, and HOS test. High correlations of sperm viabilities were found among flow cytometry, epifluorescence evaluation, HOS test (p<0.01) in fresh semen. Especially, sperm viability assessed by HOS test was highly correlated with viability by flow cytometry in all the ratios of live and dead spermatozoa, 1:0, 1:1 and 1:3 (p<0.01). The viability evaluated by ME were significantly correlated with that by flow cytometry in ratios of 1:0 and 1:3 (p<0.05) however, there was no significance in ratio of 1:1. The viability evaluated by C/p were highly correlated with that by flow cytometry in ratio of 1:0 and 1:1 (p<0.01) and significantly correlated in ratio of 1:3 (p<0.05). In frozen-thawed spermatozoa, the viability determined by HOS test was considerably correlated with that by flow cytometry (p<0.01). There was significant correlation between the viabilities by ME and by flow cytometry (p<0.05). But the viability evaluated by CFDA/PI was not correlated with viability by flow cytometry. The result from this study validate the use of flow cytometry as a precise method for assessing the viability of fresh and frozen-thawed dog spermatozoa.

호중구 탐식작용 측정에 있어서 유체 세포측정법 (Flow Cytometry)이용에 대한 평가

우준희,장선호,이희발,최강원 대한내과학회 1992 대한내과학회지 Vol.42 No.1

호중구 탐식 기능 측정에 있어 고식적인 면역형광 현미경 측정과 고속으로 각각의 세포에 대한 분석이 가능한 유체 세포측정법(flow cytometry)을 이용한 방법을 건강한 의과대학생 9명을 대상으로 측정하여 다음과 같은 결과를 얻었다. 면역형광 현미경으로 측정한 탐식 호중구의 비율과 유체 세포측정법을 이용하여 측정된 비율간에는 γ=0.76으로 유의한 상관관계가 있었다(p<0.05). 따라서 유체 세포측정법(flow cytometry)에 의한 호중구 탐식기능 측정은 신속하고 정확하게 각각의 세포 특성에 대한 분석을 가능케하는 방법으로, 탐식작용은 물론 종양세포분석, 면역세포학등 다른 여러 부분에도 이용이 가능할 것으로 생각된다. Neutrophils are one of the essential components of the host defense system. The importance of adequate phagocyte function has been demonstrated by the association of specific celluar defects with increased susceptibility to infection. Phagocytic activity of neutrophils was evaluated using flow cytometry, which allowed measurements to be made on a single-cell basis at high speed. Neutrophils collected by a Ficoll-Hypaque density gradient centrifugation were incubated with fluorescent microspheres of uniform size in pooled human plasma and washed in buffer to remove the extracellular beads. Phagocytic activity was also evaluated by fluorescence microscopy and the results were compared to those of flow cytometry. High degree of correlation was observed between the two methods. Flow cytometry provided additional informations: percentage of phagocytic cells and the distribution of intracellular fluorescent microspheres per cell. Thus the advantages of speed, elimination of subjective interpretation and the ability to provide informations on the cell population indicate that flow cytometry has a unique role in the study of phagocytosis.

Flow Cytometry를 이용한 백혈병 세포내 항원의 측정

이정운,남정현,권오헌 大韓血液學會 1995 大韓血液學會誌 Vol.30 No.3

HLA 교차시험에서 AHG-CDC의 유세포 측정: 예비연구

원동일 대한수혈학회 2010 大韓輸血學會誌 Vol.21 No.2

Background: For HLA crossmatch in organ transplantation, complement-dependent cytotoxicity (CDC) is a very useful methodology to detect donor-specific HLA antibodies and their complement fixing ability. In this preliminary pilot study, we investigated whether dead cells induced in anti-human globulin (AHG)-augmented CDC (AHG-CDC) reaction could be measured by flow cytometry (‘FC AHG-CDC’). Methods: This FC AHG-CDC measured the percentage of dead cells (% dead cells) after 7-aminoactinomycin D staining using 3 color flow cytometry. A total 45 flow cytometry crossmatch (FCXM) cases of 12 positives and 33 negatives were further tested using this FC AHG-CDC. Results: The % dead cells of FC AHG-CDC was significantly correlated with the mean fluorescent intensity ratio of FCXM (T cells: r=0.613, P=7.45×10-6; B cells: r=0.404, P=0.006). The positivity rate of FC AHG-CDC among FCXM positive cases was relatively high: 80% (8/10) for T cells and 75% (9/12) for B cells. The negativity rate of FC AHG-CDC among FCXM negative cases was 100% (35/35) for T cells and 91% (30/33)for B cells. Conclusion: In this pilot study, FC AHG-CDC could yield quantitative values, % dead cells, which was proportional to the level of complement-fixing cytotoxic antibodies against T and B cells, respectively, even without physical separation of cells or serial dilution of serum. (Korean J Blood Transfus 2010;21:122-131) 배경: 장기이식을 위한 HLA 교차시험에서 보체의존세포독성법(CDC)은 기증자특이 HLA 항체의 존재와 보체고정 능력을 평가하는데 매우유용한 방법이다. 본 예비연구에서는 anti-human globulin (AHG)-증강 CDC (AHG-CDC) 반응 유발후 죽은 세포를 유세포분석법(flow cytometry, FC)으로 측정할 수 있는지 조사하였다(‘FC AHGCDC’). 방법: 본 FC AHG-CDC는 삼색상 유세포분석법으로서, 7-aminoactinomycin D로 염색하여 죽은세포 비율(% dead cells)을 측정하였다. flow cytometry crossmatch (FCXM) 총 45예(양성 12예, 음성 33예)에서 FC AHG-CDC를 시행하여 비교하였다. 결과: FC AHG-CDC의 % dead cells는 FCXM의mean fluorescent intensity ratio와 유의한 상관관계가 있었다(T세포: r=0.613, P=7.45×10−6; B세포:r=0.404, P=0.006). FCXM 양성예중 FC AHG-CDC 양성률은 T, B세포 각각 80% (8/10), 75% (9/12)로비교적 높았다. FCXM 음성예중 FC AHG-CDC 음성률은 T, B세포 각각 100% (35/35), 91%(30/33)이었다. 결론: 본 예비연구에서, FC AHG-CDC는 T와 B 세포의 물리적 분리와 혈청 배수 희석 없이도 각세포에 대한 보체고정 세포독성 항체의 농도에비례하는 정량적인 측정치인 % dead cells을 얻을수 있었다.

Recent advances in miniaturized microfluidic flow cytometry for clinical use

Chung, Taek Dong,Kim, Hee Chan WILEY-VCH Verlag 2007 Electrophoresis Vol.28 No.24

<P>This article provides an overview of recent research achievements in miniaturized flow cytometry. The review focuses on chip-based microfluidic flow cytometers, classified by cell transport method, detection technology, and biomedical application. By harnessing numerous ideas and cutting-edge microfabrication technologies, microfluidic flow cytometry benefits from ever-increasing functionalities and the performance levels achieved make it an attractive biomedical research and clinical tool. In this article, we briefly describe an update of recent developments that combine novel microfluidic characteristics and flow cytometry on chips that meet biomedical needs.</P>

2-Bromopropane에 의한 유발된 Sprague-Dawley 랫트의 고환위축의 병리학적 관찰 및 Flow Cytometry를 이용한 검사

손화영,강부현,조성환,차신우,노정구 한국독성학회 1998 Toxicological Research Vol.14 No.2

This study was carried out to evaluate the testicular toxicity of 2-bromopropane (2-BP), which recently caused occupational intoxication on the reproductive and hematopoietic system in Koreans, using light microscopy, immunohistochemistry and flow cytometry. 10 weeks old male Sprague-Dawley (SD) rats were treated with 0.5 g/㎏/day of 2-BP orally for 8 consecutive weeks. The testes of the rats were vascularly perfused with Karnovsky's solution or immersed in Bouin's solution, embedded in plastic and evaluated with light microscopy. And relative proportions of haploid, diploid, and tetra-ploid states of DNA ploidy in the testicular cell suspensions of the SD rats were examined by flow cytometry. 2-BP induced severe testicular atrophy, depletion and degeneration of spermatogonia, spermatocytes, and spermatids and mild hyperplasia of Leydig cells without significant morphological changes. The Leydig cell hyperplasia was confirmed by immunohistochemistry using proliferating cell nuclear antigen (PCNA). The immunopositive cells against PCNA were observed in the nuclei oj some interstitial cells. Relative proportions of haploid states of DNA ploidy decreased in the atrophic testicular cell suspensions comparing with those of the control. In conclusion, 2-BP induced testicular atrophy with Leydig cell hyperplasia as examined by histopathology, immunohistochemistry and DNA flow cytometry.

OVCAR-3 세포주에서 Annexin V, Propium Iodide와 Cytokeratin 염색에 의한 고사세포의 측정

송민경 ( Song Min Gyeong ),권문기 ( Kwon Mun Gi ),이정웅 ( Lee Jeong Ung ),최예훈 ( Choi Ye Hoon ),김태우 ( Kim Tae U ),류기성 ( Ryu Ki Sung ),나종구 ( Rha Jong Gu ),한구택 ( Han Gu Taeg ) 대한산부인과학회 2003 Obstetrics & Gynecology Science Vol.46 No.7

목적 : 세포사의 한 종류인 고사 (apoptosis)는 인체의 항상성 (homeostasis)을 유지하고 균형을 유지하기 위한 세포사의 다양한 과정의 하나이다. 최근에는 이러한 고사의 정도를 정량적으로 측정함으로써 항암화학요법제의 투여전 감수성 지표로 이용해 보려는 연구들도 진해되고 있다. 따라서 본 연구자들은 OVCAR-3 난소암 세포주에서 taxol을 투여한 후 세포주기 분획의 변화 및 G_0G_1 전분기 분획의 출현과 annexin V와 PI 또는 cytokeratin의 염색에 따른 고사 세포들을 flow cytometry로 분석하고 감별 측정함으로써 항암 요법제의 투여와 관련된 세포사의 기전을 이해하고 고사 분획의 측정이 항암화학 요법제의 투여전 약제 감수성의 지표로 이용가능성이 있는지를 알아보고자 본 연구를 계획하였다. 연구 방법 : OVCAR-3 난소암 세포주에서 대조군을 포함하여 20 nM 또는 30 nM의 taxol을 가하고 24시간과 48시간 동안 배양한 후 세포들을 수확하였다. 실험 (1). 1x10^5개의 세포가 들어 있는 시험관에 annexin V-FITC와 propidium iodide (이하 PI)를 가한 후 배양하고 이와 병행하여 다른 하나의 시험관의 세포에는 DNA 염색을 위해 PI를 가하고 배양하였다. 실험 (2). 1x10^5개의 세포가 들어 있는 시험관에 annexin V-FITC와 cytokeratin (CAM5.2와 MMF116)을 가하고 배양한 후에 1% paraformaldehyde 용액과 100% methanol 용액으로 세포를 고정하고 침투시켰으며, 다음에 GAM IgG_1-PE 또는 GAM IgG_2a-PE 항체를 가하고 다시 배양하였으며, 이어서 PI에 의한 DNA의 염색을 실시하였다. 실험 (1)과 (2)에서 염색된 세포들을 표준 FACScan flow cytometer로 분석하였다. 결과 : (1) OVCAR-3 세포주에서 20 nM과 30 nM의 taxol의 투여로 G_2M 분기의 정지되었으며, 24시간과 48시간으로 배양 시간이 증가함에 따라 그 정도는 급격히 증가하였으며, 그 증가의 정도는 20 nM의 taxol 투여에 비하여 30 nM의 taxol 투여의 경우에 현저히 증가되었다. (2) DNA 분절화와 고사를 뜻하는 G_0G_1 전분기 분획이 대조군으로부터 24시간 및 48시간으로 taxol과의 배양 시간이 증가됨에 따라 증가되었다. (3) Annexin V-FITC 양성과 PI 음성으로 표현된 조기 고사 세포의 출현은 대조군에 비하여 taxol의 투여 후 24시간 동안 배양한 경우로부터 배양 시간이 48시간으로 증가됨에 따라 점차 증가하였다. (4) 30 nM의 taxol을 투여한 후 조기 고사 세포를 감별 측정하기 위해 annexin V-FITC와 cytokeratin (CAM5.2 및 MNF116)의 염색과 연속적으로 DNA 함량을 위한 PI 염색을 동시에 실시한 연구에서 조기 고사 세포 (annexin V 양성 및 cytokeratin 음성)의 출현은 대조군으로부터 24시간 및 48시간 동안 배양 시간이 증가됨에 따라 증가되었으나 그 증가 정도는 20 nM의 taxol을 투여하고 annexin V-FITC와 PI의 염색으로 측정된 결과에 비하여 낮았다. 결론 : 본 연구자들이 PI에 의한 DNA의 염색과 병행하여 annexin V와 PI에 의한 고사의 관찰과 annexin V, cytokeratin 및 PI에 의한 DNA의 연속적인 동시 염색에서 얻은 결과들이 거의 서로 일치함을 관찰한 것은 항암제 투여후 flow cytometry로 측정된 고사가 항암제 감수성의 간접적인 지표로서 이용 가능성을 확인한 결과들이라고 생각되나 이의 확인을 위한 연구가 향후 더 진행되어야 할 것으로 사료되었다. Objective : This study was designed to estimate the chemosensitivity by a quantitative evaluation of the apoptotic cell fractions using flow cytometry. Methods : The OVCAR-3 cells were exposed to 20 nM or 30 nM taxol for 0 (control), 24 and 48 hours, then removed the taxol contained media, and cultured further with fresh media without taxol. (1) Fluorescin isothiocyanate-conjugated Annexin V (Annexin V-FTTC) and propidium iodide (PI) were added to one test tube to detect the apoptotic cell fractions and at the same time, PI was added to the other tube to stain the DNA. (2) Annexin V-FITC and cytokeratin (clone CAM5.2 and MNF116) were added to the test tube. They were fixed and permeabilized with 1% paraformaldehyde solution and 100% methanol. They were then incubated with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (GAM IgG_1-PE or GAM IgG_2a-PE) and sequentially stained with PI for DNA. All the stained cells were analyzed by a FACScan flow cytometer. Results : (1) After treatment of 20 nM or 30 nM of taxol, G_2M arrest was observed in both of treatment groups, which increased with time. (2) The G_0G_1 sub-fraction indicative of apoptosis increased with increase of culturing time from 24 hrs to 48 hrs. (3) The early apoptotic cell fraction with positive annexin V-FITC and negative PI increased with increase of culturing time. (4) In cells stained sequentailly with annexin V-FITC, cytokeratin (CAM5.2 and MNF116), and PI after 30 nM taxol treatment, the early apoptotic cell fractions increased with increase of culturing time. However, their extent was somewhat lower than those observed by positive annexin V-FITC and negative PI in cells treated with 20 nM of taxol. Conclusion : The results of sequential stainings with annexin V-FITC, cytokeratin, and PI were consistent with the those of annexin V-FITC and PI with parallel DNA staining. Our results suggested that the level of apoptosis detected by flow cytometry could be a marker of chemosensitivity which could select the sensitive anti-cancer agents before administration to gynecologic cancer patients.

유세포분석법으로 조사한 Orientia tsutusgamushi Boryong주의 항균제 감수성

김은실,김미경,이혜명,정문현,이진수,박재은,강재승 대한감염학회 2008 Infection and Chemotherapy Vol.40 No.4

Background : Scrub typhus, an infectious disease caused by Orientia tsutsugamushi, is endemic in Korea. With the introduction of tetracycline and chloramphenicol in clinical practice, the mortality due to scrub typhus has markedly decreased. In 1995, scrub typhus poorly responsive to doxycycline was reported in Thailand; the need for safe antibiotics for the treatment of scrub typhus acquired during pregnancy or for children is emerging; also, broader spectrum antibiotics having anti-Orientia activity may be preferred for empirical therapy of enteric fever syndrome and for complicated scrub typhus. The anti-Orientia activities of various antibiotics, including recently licensed antibiotics, were investigated by flow cytometry. Materials and Methods : O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi. Then the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). A concentration at which no further decrease in growth index occurred was defined as the minimal inhibitory concentration (MIC). Microbial susceptibilities to the following antibiotics were measured: quinupristin-dalfopristin (Synercid), levofloxacin, ciprofloxacin, moxifloxacin, metronidazole, linezolid, clindamycin, chloramphenicol, doxycycline, azithromycin, and rifampin. Results : Considering the usual serum concentrations of rifampin (MIC=0.025-0.05 µg/mL), azithromycin (MIC=0.05-0.5 µg/mL) and doxycycline (MIC=0.05-0.1 µg/mL), these antibiotics exhibited very low MICs. Synercid (MIC=0.2-1.0 µg/mL), clindamycin (MIC=1.0 µg/mL) and chloramphenicol (MIC=1-2 µg/mL) exhibited moderately low MICs; moxifloxacin (MIC=8 µg/mL), ciprofloxacin (MIC=25.6 µg/mL or more) and levofloxacin (MIC=30 µg/mL) exhibited relatively high MICs; and cefotaxime (MIC>50 µg/mL), metronidazole (MIC>30 µg/mL) and linezolid (>30 µg/mL) exhibited high MICs. Conclusions : Among the new antibiotics, none was superior to doxycycline, azithromycin or rifampin with respect to anti-Orientia activity. Synercid, clindamycin, and moxifloxacin may show moderate therapeutic efficacies in human. Background : Scrub typhus, an infectious disease caused by Orientia tsutsugamushi, is endemic in Korea. With the introduction of tetracycline and chloramphenicol in clinical practice, the mortality due to scrub typhus has markedly decreased. In 1995, scrub typhus poorly responsive to doxycycline was reported in Thailand; the need for safe antibiotics for the treatment of scrub typhus acquired during pregnancy or for children is emerging; also, broader spectrum antibiotics having anti-Orientia activity may be preferred for empirical therapy of enteric fever syndrome and for complicated scrub typhus. The anti-Orientia activities of various antibiotics, including recently licensed antibiotics, were investigated by flow cytometry. Materials and Methods : O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi. Then the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). A concentration at which no further decrease in growth index occurred was defined as the minimal inhibitory concentration (MIC). Microbial susceptibilities to the following antibiotics were measured: quinupristin-dalfopristin (Synercid), levofloxacin, ciprofloxacin, moxifloxacin, metronidazole, linezolid, clindamycin, chloramphenicol, doxycycline, azithromycin, and rifampin. Results : Considering the usual serum concentrations of rifampin (MIC=0.025-0.05 µg/mL), azithromycin (MIC=0.05-0.5 µg/mL) and doxycycline (MIC=0.05-0.1 µg/mL), these antibiotics exhibited very low MICs. Synercid (MIC=0.2-1.0 µg/mL), clindamycin (MIC=1.0 µg/mL) and chloramphenicol (MIC=1-2 µg/mL) exhibited moderately low MICs; moxifloxacin (MIC=8 µg/mL), ciprofloxacin (MIC=25.6 µg/mL or more) and levofloxacin (MIC=30 µg/mL) exhibited relatively high MICs; and cefotaxime (MIC>50 µg/mL), metronidazole (MIC>30 µg/mL) and linezolid (>30 µg/mL) exhibited high MICs. Conclusions : Among the new antibiotics, none was superior to doxycycline, azithromycin or rifampin with respect to anti-Orientia activity. Synercid, clindamycin, and moxifloxacin may show moderate therapeutic efficacies in human.

HIV 감염자에서 flow cytometry를 이용한 p24 양성 CD4+ T 림프구의 측정

김영봉,조영걸,최병선,장영식,양재명,신영오 대한바이러스학회 1994 Journal of Bacteriology and Virology Vol.24 No.1

The identification of factors that correlate with progression of human immunodificiency virus (HIV) infection is important for prognosis and monitoring of therapy. We tried flow cytometric method for detection of HIV p24 antigen in CD4+ cells. Lymphocytes harvested from HIV infected persons were treated with a methanol:aceton fixative solution for giving permeability and followed by binding of monoclonal antibody specific for HIV p24 antigen. After labeling with a fluorescein isothiocyanate-conjugated F(ab) fragments of goat anti-mouse immunoglobulin antibody, p24 antigen positive CD4+ cells were counted using flow cytometry. We followed 40 HIV infected persons during the period of Sept. 1992 and Sept. 1993. The pro- portion of p24 antigention of CD4+ cells(Y) 3 months before and after the test (Y=-4.59X+ 44.45). On the other hand there was less correlation [r=-0.356(p=0.046)] with absolute CD4 +lymphocyte count which was directly linked to clinical stage. This results suggest that flow cytometry can be used as a rapid, sensitive and quantitative assay system for detection of viral replication status of HIV-seropositive persons and it could be the most accurate prognostic marker for HIV disease.

Flow cytometry를 이용한 체강액의 DNA 분석

최윤미,지현숙 대한임상병리학회 1992 대한임상병리학회지 Vol.12 No.2