What is the best way to verify that cells die by excess autophagy?

Autophagy (or autophagocytosis) (from the Greek auto-, "self" and phagein, "to eat"), is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. The breakdown of cellular components promotes cellular survival during starvation by maintaining cellular energy levels. Autophagy allows the degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled.

There are three different forms of autophagy that are commonly described; macroautophagy, microautophagy and chaperone-mediated autophagy.

In the context of disease, autophagy has been seen as an adaptive response to stress which promotes survival, whereas in other cases it appears to promote cell death and morbidity.

It is well known that cells that are Propidium Iodide (PI) -ve and annexin V (AV) -ve are alive cells; PI-ve AV +ve are early apoptotic cells; PI+ve AV+ve late apoptotic cells and PI+ve AV-ve are damaged cells (necrotic cells). So, does this apply for the staining with LIVE/DEAD and AV?

Attached is an example of staining with LIVE/DEAD and AV.

Is there a signaling pathway or a particular signal induced by arrest between S phase and mitosis (cells don't divide after synthesizing new DNA) that initiates cell death? Is this death necessarily mediated by caspases?

Hi all

I use a compound to induce cell death and found it could be enhanced by zVAD-FMK. So we could exclude the apoptosis and proptosis and considered it might be necroptosis. However, necrostatin-1 had no effect on it. 

According to current cell death types we knew, can I confirm it is autophagy if it can be inhibited by PI3K inhibitor? Any other experiment I should do to confirm it?

Thank you so much.

I treated an adherent neuroblastoma cell line population with 6 candidate novel compounds, and a DMSO-control, for 24 hours, and conducted a TUNEL assay to quantify apoptotic figures. All of the results, including DMSO came out negative. Those compounds had previously demonstrated reproducible cytotoxic effects in viability assays. I am wondering as to why they may have come out negative? Sure, there are many ways other than apoptosis that the drugs may be causing cytotoxic effects, but in a normal population of neuroblastoma cells are a few not usually undergoing apoptosis anyway? My TUNEL protocol was verified by two independent researchers, and repeated twice, with the same results. One thing I did notice was that the cells were very sparse, probably because I didn't add a chemical to increase cell adherence to the cover slips on which they were incubated. Could it be that the sparsity of the cells caused too much background noise for any apoptotic cells to be seen? At 20x zoom, the number of cells in the field of view varied between 6 and 150, but all were negative for apoptosis.

Other cells had previously been tested for proliferation using phosphohistone-H3 staining, and proliferation for all but one of the drug-treated cells as well as DMSO (control) cells was around 5%. One of the drugs caused proliferation to increase to 20%.

Any help in interpreting these results would be greatly appreciated.

I tried to see the autophagy formation in the Rapamycin treated cells by using autophagy dye.Did any one tried by measuring fluorescence of autophagy dye added to the cells after induction?

I use a stimulator of apoptosis to 300 doses. When using 900 doses causes increased apoptosis but also a decrease in necrosis (dead cells and too). I could not justify this decrease I think unreasonable. If someone has read something about him I would be grateful indicate. Thank you for all

In a normal cell, which one of the below mentioned mechanisms of cell death occurs first?

Apoptosis or autophagy or necrosis

Apoptosis is a programed cell death. It occurs in multi-cellular organisms to regulates various physiological function. There are mainly two kinds of apoptosis namely extrinsic apoptotic pathway.