We treated Lo2 liver cells with a compound for 24 hrs and the cells turned like attached image. There are multiple holes in the cells. So, is that a consequence of apoptosis or the forming of lipid droplets? Anyone have any experience of this?
Thank you for your answers, and they really give us lots inspiration. As Dominique mentioned, we stained cells with trypan blue, as shown in the attached image. We saw only few positive staining in the attached cells. Only detached cells shown the blue color, which means the cells are still alive. So, we are trying to withdraw the drug and see whether this phenomena can be reversed.
Regards!
Think about Methuosis
http://molecular-cancer.biomedcentral.com/articles/10.1186/1476-4598-10-69
Increase number of holes after the drug treatment inside the cells could be autophagic vacuoles, as induced autophagy can also leads to cell death. To clarify this, you can look for caspase 3 (apoptotic marker) and LC-3 (autophagy marker) levels in you drug treated lysates ,and compare it with the untreated controls.
it seems like accumulation of autophagic vacuole that could be a morphological characteristic of apoptosis / autophagic cell death .
what surprises me is that your cells in fact remain attached to the bottom of culture flask and that their external plasma membranes appear to be almost intact. Have you tried to determine whether or not your cells are dead or alive (trypan blue stain, or Lactate dehydrogenase release). What is the time course of your morphological changes over a 24-48h period ? Are these morphological changes reversible when you washed the medium to add a new medium without drug ? These simple experiments can perhaps give you some answers
Sincerely your's
D Duval
Hi Eric,
Methuosis is very interesting! I would first rule out the vacuoles for lipid droplets using oil red O or Nile red staining.
Sathish
Hi Eric,
What you see is quite similar to our recent work (https://bmccellbiol.biomedcentral.com/articles/10.1186/s12860-015-0050-z). In this article you can find some methods to characterize origination and properties of vacuoles. As well, you can refer to this review about types of cell death-associated vacuolization: http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=10150
Hope this helps.
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