I am observing cell death upon treatment of a a certain drug in A549 cells (lung cancer adherent). But I could not make out whether it is apoptosis, necrosis or autophagy. The cells are rounded and with flattened morphology with dendrites like projections. So can any one help me differentiate them visually, or using western blot?
Hello. You will use May-Grünwald- Giemsa for necrosis. I will send one link for you. This information is more useful for you. Any way advance all the best for your carrier.
http://ajplung.physiology.org/content/281/6/L1392.long
better to use EtBr and Acridine orange staining for this. You can easily differentiate cells under florescence microscope showing different color indicate necrosis, apoptosis as well as early apoptosis
better to use EtBr and Acridine orange staining for this. You can easily differentiate cells under florescence microscope showing different color indicate necrosis, apoptosis as well as early apoptosis. You can check levels of apoptotic and anti-apoptotic proteins using western blotting to confirm.
In order to know how are you triggering apoptosis and where you should start looking you can do 2 things:
1- To do a time-lapse microscopy (video) for 48-72 hours based on the drug that you're using. Take movie frames every 15-20 minutes in 4-5 different spots on the plate (both in control and treated wells) - you can do it in either 6 or 12 well plates.
2- There are different apoptotic and apoptosis pathway markers that you can use in Western Blot. Caspase-3, PARP, Bcl-2, Bcl-x, Bcl-A1, Livin, Survivin, CIAP-1, CIAP-2, BID, and BIM. You can use the same cell lysate for all of them, however, if you want to do a Cytochrome C then the same lysate will not work.
The movie + these markers will give you insights of whats going on and where you should look really in order to understand the underlying cell death mechanism.
To differentiate between different cell death programs then Arjan's answer does explained that :).
Apoptosis can separate from necrosis and autophagy by DAPI staining or DNA ladder assay. Flow cytometry is very useful , i use tunel assay for apoptosis because it specific for apoptosis. For western blot PARP 2 band is marker of apoptosis but you can use proteome profiler from R&D for finding possible apoptosis pathway. Necrosis can use RIP1 kinase or BNIP 3 for western blot. Autophagy use beclin 1 for western blot.
Hi,
You can try LC3 and P62 antibodies to check autophagy using western blot. Atg5 and Beclin 1 antibodies help as well.
Hello everyone,
I need to differentiate necrosis, apoptosis and autophagy in tissue. Is there any way of doing this with paraffin-embedded tissue?
Thank you in advance.
- Badges
- Science method