Suppose I've given u two samples in which one sample contains live cell and other sample contains dead mammalian cells (Example CHO), Since both the samples are not labelled, I'ld like to know which sample is live cells and which sample is dead cells.
One method i know is :
1) Add trypan blue and observe under microscope, So that live cells appear blue whereas dead cells appear transparent (i.e. no color).
2) Turbidity of live cells increases with time whereas dead cells remain same.
I'ld like to know what all other methods are there to distinguish live cells from dead cells ?
Hello Vikram Kumar
I would like to just make a correction in your statement about trypan blue. Trypan blue dye will be excluded from membrane-intact live cells but can enter and concentrate in membrane-compromised dead cells, rendering the dead cells dark blue.
You can use cell-impermeant DNA binding dye, such as propidium iodide. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell where it will bind to the DNA and become fluorescent. The dead cells therefore will be positive, and the live cells will be negative.
You can also use the live/dead fixable stains, also known as amine reactive dyes. This also works using the principle of cell membrane integrity, where amines on the outside of a living cell with react with the amine-reactive dye and have a dim fluorescence, while a dead cell with a compromised membrane will allow the dye into the cell where it will react with amines throughout the cell resulting in a bright fluorescence.
Best.
Hi,
Go for passaging, live cells will propagate and dead cells will not.
Hello Vikram Kumar,
Quantification of a number of live and dead cells is an indispensable tool in cell biology research. Live/Dead Cell Viability Assay Kit, provides a two-color fluorescence method that is based on the simultaneous determination of live and dead cells using two different dyes. Live-cell dye easily penetrates intact, live cells and intracellular esterase hydrolyzes the dye to produce a hydrophilic, strongly fluorescent compound that is retained in the cell cytoplasm which can be measured at Ex/Em = 485/530 nm. Dead cell dye enters damaged cell membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acid, thereby producing a bright red fluorescence (Ex/Em = 495/635 nm) in dead cells. This assay kit provides an easy-to-use, non-radioactive, histological, and FACS-based method for measuring cell proliferation, cell viability, chemotaxis, cytotoxicity, and apoptosis.
Also, a healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell where it will bind to the DNA and become fluorescent.
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