Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio.
Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio.
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使用流式细胞术测定斑马鱼斑马鱼精子浓度并同时分析精子质膜完整性。
DOI：
10.1002/cyto.a.22796
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Tiersch,TerrenceR
中科院分区：
文献类型：
--
作者：
Yang,Huiping;Daly,Jonathan;Tiersch,TerrenceR
为了确保冷冻保存和体外受精方案的一致性和可重复性，需要控制精子浓度。精子浓度的测定传统上用计数室（例如，血细胞计数器），或最近使用分光光度计。对于小型生物医学模式鱼，精子样品的可用性仅限于微升，因此需要开发快速准确的浓度测定方法，同时最大限度地减少样品使用。在这项研究中，开发了一种新的方法，用于使用流式细胞仪（Accuri C6，BD Biosciences，圣何塞，CA）测定精子浓度，并在斑马鱼精子中使用SYBR®−14和碘化丙啶（PI）进行荧光染色后同时测量精子膜完整性。目的是开发一种通过流式细胞术同时测定精子质量和数量的方案。目的是（1）确定样品体积的影响（250和500 µl）和分析体积（10和50 µl）对使用标准容量验证微珠进行颗粒计数的准确性;（2）确定流式细胞术可测量的精子浓度的有效范围;（3）测试精子浓度测量的精密度和重现性;以及（4）通过与用血细胞计数器和显微分光光度计的测量进行比较来验证流式细胞术方法。250和500 µl的样品体积和10和50 µl的分析体积不影响微珠计数，工厂设定的流速为“中等”或“快速”，并且在1 × 103−1 × 107个细胞/ml的浓度范围内保持精密度和准确度。本研究中开发的方法与传统方法如血细胞计数器或显微分光光度计相当。本研究提供了一种高效、准确、快速的方法，使用流式细胞术测定精子浓度，同时提供精子膜完整性的评估。这些方法可以减少数量评估所需的时间，并最大限度地利用有价值的精子样本。© 2015国际细胞计数促进学会
Control of sperm concentration is required to ensure consistent and reproducible results for cryopreservation andin vitrofertilization protocols. Determination of sperm concentration is traditionally performed with a counting chamber (e.g., hemocytometer), or more recently with a spectrophotometer. For small‐sized biomedical model fishes, the availability of sperm sample is limited to microliters, so it is desirable to develop fast and accurate approaches for concentration determination that also minimize sample use. In this study, a new approach was developed for sperm concentration determination using a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) with simultaneous measurement of sperm membrane integrity after fluorescent staining with SYBR®−14 and propidium iodide (PI) in sperm from ZebrafishDanio rerio. The goal was to develop a protocol for simultaneous determination of sperm quality and quantity by flow cytometry. The objectives were to (1) determine the effects of sample volume (250 and 500 µl) and analysis volume (10 and 50 µl) on the accuracy of particle counting using standard volumetric validation beads; (2) identify the effective range of sperm concentrations that flow cytometry can measure; (3) test the precision and reproducibility of the sperm concentration measurements; and (4) verify the flow cytometry approach by comparison with measurement with a hemocytometer and a microspectrophotometer. Sample volumes of 250 and 500 µl and analysis volumes of 10 and 50 µl did not affect bead count with the factory‐set flow rates of “medium” or “fast,” and the precision and accuracy was retained across a concentration range of 1 × 103−1 × 107cells/ml. The approach developed in this study was comparable to traditional methodologies such as hemocytometer or microspectrophotometer. This study provides an efficient, accurate, and rapid method for determination of sperm concentration using flow cytometry while providing simultaneous assessment of sperm membrane integrity. Such approaches can reduce the time needed for quantity assessment and maximize the use of valuable sperm samples. © 2015 International Society for Advancement of Cytometry
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影响因子：
64.5
作者：
Koreaki Ito;Takashi Sato;T. Yura
通讯作者：
T. Yura
N-亚硝基乙胺依赖细胞色素 P-450 形成乙烯。
DOI：
--
发表时间：
1988
期刊：
Drug metabolism and disposition: the biological fate of chemicals
影响因子：
--
作者：
Ding,XX;Coon,MJ
通讯作者：
Coon,MJ
影响因子：
4.8
作者：
K. Kita;K. Konishi;Y. Anraku
通讯作者：
Y. Anraku
缺乏疏水性 NH2 末端片段的酒精诱导型细胞色素 P-450IIE1 保留了催化活性，并且在大肠杆菌中表达时呈膜结合状态。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Larson,JR;Coon,MJ;Porter,TD
通讯作者：
Porter,TD
使用铜诱导型 CUP1 启动子在酿酒酵母中表达酒精诱导型兔肝细胞色素 P-450 3a (P-450IIE1)。
影响因子：
3.1
作者：
Fujita,VS;Thiele,DJ;Coon,MJ
通讯作者：
Coon,MJ