Hi there,
Could anyone recommend a cheap and easy way to assay for cell death (probably apoptosis). When our gene of interest is transfected into cells, there appears to be a lot of cell death compared to cells transfected with empty vector or an irrelevant gene.
So am wondering:-
a) do I need to collect the cells that are floating in the media to analyse this effect
b) should I collect only the cells that remain attached to the plates
c) collect all cells (floating + attached) and assay for cell death?
This would be an end-point assay
Thanks in advance!
Satish
Satish Patel
If you would like to study the cytotoxicity of this plasmid, I suggest to use MTT, neutral red assay or annexin/PI as they are sensitive in detecting cytotoxic events and at the same time they are cheap.
PI: regarding the type of cells, in the mentioned assays you only detect the live cells or those in the last step of apoptosis, but if you would like to use both the live and dead one, trypan blue exclusion assay would be helpful.
Cell titer-glo is not the cheapest, but it allows you to just add it to the cells and analyze, so it would take into account floating cells as well.
A resazurin assay is a cheaper but potentially less sensitive way to do this.
If you just have a few samples you could always just trypsinize the cells and count with Trypan blue as well. In that case I would probably collect the floating cells as well and check via trypan blue how many of them are alive.
Both of these just measure how many cells are alive, they don't offer any insight into apoptosis, or difference in proliferation vs. death.
There are different ways depending on which stage of apoptosis you are looking for, I have used a few in the past :
- Annexin staining - an earlier marker for apoptosis, can be used for microscopy or flow cytometry
- Caspase 3 activation - can be used for western blot to assess activation of apoptosis pathway
- Propidium iodide - Fluorescent DNA dye that can only enter dying cells. Can be used for fluorescent microscopy and if combined with flow cytometry, it can further reveal DNA fragmentation.
- TUNEL assay - can be used for microscopy and flow cytometry to assess DNA fragmentation. Commercial kits are widely available.
- MTT and LDH - two rapid but rather crude method to assess cell death, not really specific to apoptosis. Suitable for high-throughput screening.
- GFP leakage - simply transfect the cells with a bright GFP construct. If the cells are dying, GFP can leak to medium which can be measured as a readout for cytotoxicity.
To assay for cell death, particularly apoptosis, there are several cheap and easy methods you can consider. Here are a few recommendations:
Now, addressing your specific questions:
a) If you suspect that there is significant cell death resulting in floating cells, it would be beneficial to collect the cells in the media as well. This will help you capture the full extent of cell death, including both floating and attached cells.
b) Collecting only the cells that remain attached to the plates may not provide a complete representation of cell death. However, it can still be informative to assess the level of apoptosis in the adherent cell population.
c) Collecting all cells (both floating and attached) will give you a comprehensive understanding of cell death. This approach will provide a more accurate assessment of the impact of your gene of interest on apoptosis.
Remember to validate the chosen assay method(s) and optimize the experimental conditions for your specific cell type and experimental setup. Additionally, it's essential to include appropriate controls, such as untreated cells or cells treated with known apoptotic inducers, to validate the assay results.
To assay for cell death, particularly apoptosis, there are several cheap and easy methods you can consider. Here are a few recommendations:
Now, addressing your specific questions:
a) If you suspect that there is significant cell death resulting in floating cells, it would be beneficial to collect the cells in the media as well. This will help you capture the full extent of cell death, including both floating and attached cells.
b) Collecting only the cells that remain attached to the plates may not provide a complete representation of cell death. However, it can still be informative to assess the level of apoptosis in the adherent cell population.
c) Collecting all cells (both floating and attached) will give you a comprehensive understanding of cell death. This approach will provide a more accurate assessment of the impact of your gene of interest on apoptosis.
Remember to validate the chosen assay method(s) and optimize the experimental conditions for your specific cell type and experimental setup. Additionally, it's essential to include appropriate controls, such as untreated cells or cells treated with known apoptotic inducers, to validate the assay results.
Thankyou Mohamed Khashan Stephan Spangenberg Anthony Hiu King Tsang Chandni Hussain for all your suggestions. That is really useful and gives me a strategy of how to approach it initially.
Will give trypan blue + MTT a shot initially and then refine by testing for apoptosis as you have indicated. Really helpful!!
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