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通过流细胞计测定Cryptococcus neoformans中的Ploidy水平和核DNA含量:可变性的缺点

Yun C Chang¹, Michael J Davis¹, Kyung J Kwon-Chung¹

¹Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Journal of fungi (Basel, Switzerland)

|April 26, 2024

概括

化 (PI) 染色是Cryptococcus细胞周期和化分析中最可靠的方法,尽管细胞大小存在差异. 替代染料显示不特定的染色,限制了它们在Cryptococcus DNA内容分析中的使用.

科学领域:

菌类学菌类学是指菌类学.
细胞生物学细胞生物学
生物技术是生物技术.

背景情况:

流细胞计是分析细胞种群的标准技术,包括用于化和细胞周期研究的DNA含量.
在Cryptococcus中精确的DNA含量分析对于理解真菌生物学和病原性至关重要.
传统的DNA结合染料,如酸 (PI) 是常用的,但可能会受到细胞大小变化的影响.

研究的目的:

评估化 (PI) 染色对Cryptococcus ploidy和细胞周期分析的可靠性.
评估替代DNA结合染料 (SYTOX绿色,SYBR绿色I) 以提高Cryptococcus DNA分析的准确性.
为了研究细胞大小变异对基于流细胞计的Ploidy确定Cryptococcus细胞大小变异的影响.

主要方法:

流式细胞计量用于细胞周期和Ploidy确定在Cryptococcus.
细胞被固定并染色为DNA结合的光染料,主要是化 (PI).
使用ImageStream系统分析与细胞大小相关的光强度变化.

主要成果:

酸 (PI) 染色显示光强度因Cryptococcus细胞大小的显著变化而变化,混了准确的化分析.
图像流系统成功地解释了光强度的变化,与细胞大小增加引起的性变化无关.
在Cryptococcus中,SYTOX Green和SYBR Green I展示了非特异性的核DNA染色,与它们在Saccharomyces cerevisiae中的表现不同.

结论:

尽管细胞大小变化的局限性,但化 (PI) 仍然是目前Cryptococcus细胞周期和化分析的最可靠方法.
需要进一步开发DNA结合染料或方法,以减轻由Cryptococcus细胞大小和形状的巨大变化引起的变异性.
像SYTOX Green和SYBR Green I这样的替代染料不适合在Cryptococcus中染色核DNA.

关键词:

DAPI DAPI 这是一个很好的方法.霍克斯特 33342 公司在SYBR绿色I上赛托克斯绿色绿色的流动细胞计量是流动细胞计量的方法.图像流动细胞计图像流动细胞计普洛伊迪的决定性决定.酸 (Propidium Iodide) 是一种酸的化合物.

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Flow Cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
In...