我如何为LIVE/DEAD细胞活力实验准备死细胞对照？
有两种简单的方法。一种是通过60°C放置20分钟热灭活细胞。第二种是将细胞放入70%乙醇。乙醇固定的细胞可以在冰箱中无限期储存直到使用，可达好几年。
1.离心细胞、使细胞沉淀并去除上清
2.固定细胞：在15mL含有细胞团的试管中加入10 mL70%冰乙醇，先开始逐滴加入后震荡，混合均匀。
3.在冰箱中储存直到使用。
4.快使用时，水洗两次且在选择的缓冲液中重悬。
Which species of yeast/fungi can I use with the LIVE/DEAD Yeast Viability Kit?
The LIVE/DEAD Yeast Viability Kit has been tested on the following fungi: S. cerevisiae (five different strains), Candida pseudotropicalis, Neurospora crassa and Aspergillus nidulans. A good correlation between the results obtained with the LIVE/DEAD Yeast Viability Kit and those obtained with standard plate counts was achieved with both S. cerevisiae and C. pseudotropicalis. Tests have been performed on both logarithmically growing cultures and cells that have undergone different forms of environmental stress
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How do I prepare dead cell controls for LIVE/DEAD cell viability assays?
There are two easy options. One is to heat-inactivate the cells by placing at 60 degrees C for 20 minutes. The second is to subject the cells to 70% ethanol. Alcohol-fixed cells can be stored indefinitely in the freezer until use, potentially up to several years.
Centrifuge cells, pellet, and remove supernatant.
Fix cells: Add 10 mL ice cold 70% ETOH to a 15 mL tube containing the cell pellet, adding dropwise at first while vortexing, mix well.
Store in freezer until use.
When ready to use, wash twice and resuspend in buffer of choice.
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Can FUN 1 stained cells be examined by flow cytometry?
Yes. Use a 488 nm laser line and standard FITC and PE channels for two-color detection of green (dead/metabolically inactive cells) and red (live, metabolically active cells) emission.
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Live, metabolically active fungi transport FUN 1 into vacuoles to give a red-shifted fluorescence versus green/yellow fluorescence in the nucleus and cytoplasm of dead or metabolically-inactive cells. Is this a reliable indicator of fungal viability?
No. FUN 1 accumulates into vacuoles by an unknown transport pathway, but any mutants/ recombinant cells or experimental treatments that result in a deficiency or block in vesicle-mediate transport into vacuoles may result in cells that do not have red vacuoles, even though the cells are live and metabolically active. For more information see J Microbiol Methods 78:208 (2009).
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