There are a number of stains to determine viability of protoplasts, one of them is Fluorescein Diacetate (FDA) stain that has been used often, and as an alternative there is Evan's blue stain, bromophenol blue stain, methylene blue stain and phenosafranin stain. From these stains, what is the best stain for evaluating viability of protoplasts? especially, without ambiguity of the viability of protoplasts.
Hi, Reni,
I agree with Phillip, FDA is one of the best and easy dye if you have a fluoresent microscope. BUT you have to consider that FDA also is pH -dependent if you use 488 nm exitation. You can try 440 nm You can also try DAPI wihich does not penetrate to living cells, but have a bright nuclues signal in the cell with damage cells membrane.
Evans blue you can use even without fluoresent microscope, it is ver cheap and easy.
Good luck!
Hi, Reni,
I agree with Taras and Phillip. If you use fluorescent microscopy, I recommend to combine FDA with propidium iodide (PI) for better counting of living and dead protoplasts after regeneration of cell membrane. PI penetrate damaged or not fully developped cell membrane and stained nucelli red.
Me too agree on Helena, and Phill. FDA found to be ideal for protoplast staining
The only stain I have used with protoplasts is FDA. My experience with FDA is like the other posters, it is easy to use and works well. However, for me, viability estimates were typically 15-20% higher than visual observation (e.g., spherical and chloroplasts even distributed). This doesn't mean staining is not useful, it is, but rather that staining is a proxy for viability and, thus subject to all the constraints of a proxy measurement. For example, tomato leaf mesophyll protoplasts will stain with FDA even though the protoplasts are clearly not going to survive the next 24 hrs (e.g., chloroplasts aggregated to one side and plasmalemma membrane bulging outwards). Also, the staining signal varies quantitatively between protoplasts.
You should combine a number of observations/measurements to get a more complete picture of the state of your protoplasts. For example, FDA stain shortly after isolation, calcofluor white to detect formation of new cell walls after 24-48 hours and observations of cell division frequency at 2 to 4 days and 8 to 10 days. Embedding your protoplasts in low melting temperature agarose combined with the use of an inverted microscope makes these observations easier.
There was a paper a couple of years ago that had some nice images of a time course of cotton protoplasts stained with calcofluor white (Journal of Experimental Botany, Vol. 59, No. 13, pp. 3661–3674, 2008).
Hi Phillip,
Can I get your recipe of using FDA to stain protoplast please?
Thanks.
Jin
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