At the moment I am using the Live/dead Cell Staining Kit Red Fluorescence protocol for imaging with Incell Analyzer 2200 to count dead/live cells but the thing is I am wondering whether the result is accurate or not since some of dead cells must be washed off during the washing with PBS (aspiration).
What say you?
I don't have direct experience with this method, but I agree with you. Dead cells tend to lift off the substrate. If you have a washing step, you will preferentially lose dead cells. Even without a washing step, the floating dead cells would be out of view of the imager if it is looking at the bottom of the plate, unless you centrifuge the plate first.
We've been using quite a lot of LIVE/DEAD kit in our lab and from my experience, we cant use LIVE/DEAD alone due to several limitation with this technique (if accuracy is your concern). Stiefel et al have highlighted the limitations here: Article Critical aspects of using bacterial cell viability assays wi...
Due to how SYTO9 and PI works, I interpret my LIVE/DEAD results not as "live" or "dead" cells but as "cells with intact membrane" or "cells with permeabilised membrane", respectively.
We often used LIVE/DEAD in parallel with other vitality assay like BacTiter-Glo (BTG) or RealTime-Glo (RTG) which is a luminescence based assay. We found that in some application, LIVE/DEAD results are consistence with BTG/RTG while other have huge discrepancies. You can further optimised LIVE/DEAD as per instruction in the manufacturer protocol where you may need to adjust the ratio of PI:SYTO9 depending on the type of broth media or buffer solution you are using.
All the best
Irill Ishak Thank you Irill! okay will look into it.
You can also do a Live-Dead cell assay using microscopy after staining the live cell cultures with Hoechst and PI and then imaging. Here is one of my papers to refer to for the protocol:
Bose, B., Kapoor, S., Sen, U., Nihad As, M., Chaudhury, D., & Shenoy P, S. (2020). Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage. Journal of visualized experiments: JoVE, (156), 10.3791/59924. https://doi.org/10.3791/59924
There are multiple methods using which you can assess LIVE/DEAD population. Flowcytometry is useful if you are planning to run the whole sample and get accurate count. If you are only testing on a small population then I would go with microscopy. Hoechst would be useful here as stated by Bipasha Bose
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