• Title/Summary/Keyword: flow cytometry

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Comparison of Two Fluorescent Stain Methods for Jeju Black Cattle Spermatozoa Viability Assessment by Using Flow Cytometry (제주흑우 정자 생존성 평가를 위해 flow cytometry를 사용한 두가지 형광 염색법의 비교)

  • Shin, Sang-Min;Park, Seol-Hwa;Son, Jun-Gyu;Cho, In-Cheol;Seong, Pil-Nam;Kim, Nam-Young;Woo, Jai-Hoon;Shin, Moon-Cheol;Park, Nam-Geon
    • Journal of Embryo Transfer
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    • v.32 no.3
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    • pp.221-226
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    • 2017
  • Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with $Triladyl^{(R)}$-egg yolk diluent and then was performed cryopreservation. There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

Cell Viability and Flow Cytometry Analysis of a Novel Antitumor Agent, Heptaplatin in Human Melanoma Cell Line, SK-MEL-28 (신규항암제인 Heptaplatin의 인체 흑색종세포(SK-MEL-28)에 대한 세포생존률 및 유세포 분석)

  • 최수라;명평근
    • YAKHAK HOEJI
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    • v.47 no.6
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    • pp.345-351
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    • 2003
  • Heptaplatin, cis-Malonato[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3-dioxolane]platinum(II), is a novel platinum-based antitumor agent with clinical potential against human stomach cancer and the 3rd generation of the cisplatin. This study was performed to study how cisplain, heptaplatin and sunpla which is a mixture of heptaplatin and mannitol (w: w=l : 2) affect cell viability of SK-MEL-28 human melanoma cell line. Heptaplatin ($IC_{50}$/; 95.35 $\mu$M) and sunpla ($IC_{50}$/; 10.95 11M) were less effect than cisplatin (IC $_{50}$; 10.92 $\mu$M) on the SK-MEL-28 cells. By cell cycle analysis using flow cytometry, it was identified that the cells were arrested at G2/M phase by cisplatin, heptaplatin and sunpla, and percentage of cell death group was increased according to increasing of time and concentration. These results suggest that cisplatin, heptaplatin and sunpla are a novel anticancer agent against human melanoma cell.l.

Analysis of Sexed Sperm by Flow Cytometry in Hanwoo (Korean Native Cattle)

  • Yoo, Han-Jun;Lee, Kyung-Jin;Lee, Yong-Seung;Yoon, Pil-Sang;Park, Joung-Jun;Kim, Hyeong-Cheol;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.36 no.1
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    • pp.1-6
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    • 2012
  • This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability ($84{\pm}1.15%$, $p$<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability ($79{\pm}3%$, $p$<0.05) than those of the X and Y sperm ($44.7{\pm}1.67$ and $41.7{\pm}1.2%$) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities ($24.3{\pm}1.2$ and $25.7{\pm}0.9%$, $p$<0.05) compared to that of the sorted Y sperm ($13.7{\pm}1.2%$) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher ($55.0{\pm}1.7$ and $45.0{\pm}1.5%$) than those of the sorted Y-sperm ($32.3{\pm}0.9%$, $p$<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

The exceptionally large genome of the harmful red tide dinoflagellate Cochlodinium polykrikoides Margalef (Dinophyceae): determination by flow cytometry

  • Hong, Hyun-Hee;Lee, Hyun-Gwan;Jo, Jihoon;Kim, Hye Mi;Kim, Su-Man;Park, Jae Yeon;Jeon, Chang Bum;Kang, Hyung-Sik;Park, Myung Gil;Park, Chungoo;Kim, Kwang Young
    • ALGAE
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    • v.31 no.4
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    • pp.373-378
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    • 2016
  • Cochlodinium polykrikoides is a red-tide forming dinoflagellate that causes significant worldwide impacts on aquaculture industries and the marine ecosystem. There have been extensive studies on managing and preventing C. polykrikoides blooms, but it has been difficult to identify an effective method to control the bloom development. There is also limited genome information on the molecular mechanisms involved in its various ecophysiology and metabolism processes. Thus, comprehensive genome information is required to better understand harmful algal blooms caused by C. polykrikoides. We estimated the C. polykrikoides genome size using flow cytometry, with detection of the fluorescence of DNA stained with propidium iodide (PI). The nuclear genome size of C. polykrikoides was 100.97 Gb, as calculated by comparing its mean fluorescence intensity (MFI) to the MFI of Mus musculus, which is 2.8 Gb. The exceptionally large genome size of C. polykrikoides might indicate its complex physiological and metabolic characteristics. Our optimized protocol for estimating the nuclear genome size of a dinoflagellate using flow cytometry with PI can be applied in studies of other marine organisms.

Enumeration of Weissella cibaria phage with cytometry, epifluorescence microscopy, and plaque assay (유세포분석기, 형광현미경, 용균반검사 분석을 이용한 Weissella cibaria 박테리오파지 정량분석 및 상관관계분석)

  • Park, Won Jeong;Lim, Ga-Yeon;Park, Jong-Hyun
    • Korean Journal of Food Science and Technology
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    • v.50 no.2
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    • pp.244-247
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    • 2018
  • Quantitative analysis for non-host infection bacteriophage was conducted for their enumeration. Flow cytometry and epifluorescence microscopy (EPM) were selected as counting methods. Correlation analysis was performed based on the plaque assay method on the existing host infection and consisted of Pearson correlation statistical analysis, regression analysis, and difference analysis. Analyses of 12 samples with flow cytometry and plaque assay methods showed that there was a correlation of 96.7% with Pearson correlation value r=0.967, $R^2$ 0.9352, and difference value of 1.063. Analyses of 12 samples with EPM and plaque assay methods showed that there was a correlation of 99.0% with Pearson correlation value r=0.990, $R^2$ 0.9811, and difference value of 1.605. Therefore, flow cytometry and epifluorescence microscopy would be effective for enumeration of Weissella cibaria bacteriophage with plaque assay.

Development of a New Approach to Determine the Potency of Bacille Calmette-Guérin Vaccines Using Flow Cytometry

  • Gweon, Eunjeong;Choi, Chanwoong;Kim, Jaeok;Kim, Byungkuk;Kang, Hyunkyung;Park, Taejun;Ban, Sangja;Bae, Minseok;Park, Sangjin;Jeong, Jayoung
    • Osong Public Health and Research Perspectives
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    • v.8 no.6
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    • pp.389-396
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    • 2017
  • Objectives: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette-$Gu{\acute{e}}rin$ (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. Methods: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. Results: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. Conclusion: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.

Yeast two-hybrid assay with fluorescence reporter (형광 리포터를 활용한 효모 단백질 잡종 기법 개발)

  • Park, Seong Kyun;Seo, Su Ryeon;Hwang, Byung Joon
    • Korean Journal of Microbiology
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    • v.55 no.3
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    • pp.199-205
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    • 2019
  • Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

In Vitro Production of Pig Embryos using Intracytoplasmic Injection of Flow Cytometry Sorted Boar Spermatozoa

  • Kim, Dae-Young;Hyun, Sang-Hwan;Lee, Eun-Song
    • Journal of Embryo Transfer
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    • v.23 no.4
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    • pp.275-281
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    • 2008
  • The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ($44{\sim}48\;h$ vs. $40{\sim}43\;h$). Their cleavage ($65{\sim}70%$) and blastocyst formation ($9{\sim}12%$) rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.

DNA Ploidy in Anaplastic Carcinoma of the Thyroid Gland by Image Analysis (갑상선 역형성암종의 DNA 배수성에 관한 화상분석학적 연구)

  • Lee, Ji-Shin;Lee, Min-Cheol;Park, Chang-Soo;Juhng, Sang-Woo
    • The Korean Journal of Cytopathology
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    • v.6 no.1
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    • pp.10-17
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    • 1995
  • Anaplastic carcinoma of the thyroid gland is one of the most malignant tumors. Recently, DNA ploidy measured by flow cytometry and image analysis has been suggested as an additional useful indicator of tumor behavior. Studies on the occurrence and clinical significance of DNA aneuploidy in anaplastic carcinoma of the thyroid are rare. In this study, the pattern of DNA ploidy was measured by image analysis on Papanicolaou stained slides in four cases of anaplastic carcinoma and also measured by flow cytometry using paraffin blocks in two cases. In all cases of anaplastic carcinoma, DNA aneuploidy was found by image analaysis. By flow cytometry, one case had a diploid peak and the other case had an aneuploid peak. According to the above results, we conclude that anaplastic carcinoma of the thyroid glands have a high incidence of DNA aneuploidy and image analysis using Papanicolaou stained slides is a useful method in detecting DNA aneuploidy.

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Variation of Blood T Lymphocyte Subgroups in Patients with Non- small Cell Lung Cancer

  • Wang, Wen-Jing;Tao, Zhen;Gu, Wei;Sun, Li-Hua
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.8
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    • pp.4671-4673
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    • 2013
  • Objectives: To study variation in T lymphocyte subgoups and its clinical significance in non-small cell lung cancer (NSCLC). Methods: Levels of CD3+, CD4+, CD8+, CD4+/CD8+, NK and Treg cells in peripheral blood of NSCLC cases and healthy adults were determined by flow cytometry. Results: CD3+, CD4+ and CD4+/CD8+ ratio and NK cells in NSCLCs were decreased significantly in comparison with the control group (P < 0.01), and decreased with increase in the clinical stage of NSCLC, while CD8+ cells demonstrated no significant change (P > 0.05). Treg cells were significantly more frequent than in the control group (P < 0.01), and increased with the clinical stage of NSCLC. Conclusion: The cellular immune function of the NSCLC patients is lowered. It is important to detect change of T lymphocyte subgroups by flow cytometry for the diagnosis, treatment and prognostic assessment of NSCLC patients.