Go in with a Hoechst-PI staining... that'll be more authentic than only Hoechst. For a cheaper alternative to study apoptosis, u can do simple Giemsa staining which will show nuclear degradation in case an apoptotic pathway is activated.

Couple of things. First off, I think Promega is sending out free samples of a Caspase glo assay for apoptosis, so you may want to check that out. Second, are you looking for early apoptosis, or late apoptosis events? If you trypan blue stain your apoptotic cells are they blue? What are you doing to your cells to induce apoptosis? You may be able to look into what apoptotic pathway it works through and whether you are expecting dsDNA breaks or not. If you have DAPI it of course doesn't hurt to try that first. If you see Pignotic nuclei then it is a good indication that they are apoptotic. You can also look at your cells just under namarski or something and see if you can see membrane blebbing. Obviously more expensive, but the best options would to either do a caspase assay or annexin V propidium iodide. -- Ask some companies for free samples of things. That usually works for me!

@Gunjan Yeah I have been planning to use PI along with the nuclear stains.....just not sure which is better. Apoptosis occurs in the cells for sure as i have observed so far. What i wanted to check is if necrosis also checks in. Giemsa- i had read about it-- but not in plan so far- may be i need to rethink on it.

@Shelley

Thanks for informing about the Caspase glo assay promega is giving away.

I am looking for late apoptosis events--I have been using H2O2 to induce apoptosis in cells and it causes both ssDNA and dsDNA breaks.

Apoptosis occuring is sure in my case--what can i do to see if there is necrosis also side by side.

Thank you both for your ideas.

@Shelley

I started looking in details for Annexin V-PI staining and found it mentioned that for adherent cells.. annexin v may not be a good idea.

It says---

This technique is not suitable for cells from solid tissue or adherent cells because of the damage to plasma membrane caused in the attempt to disaggregate cells to have a single cell suspension. Any procedure which affects the integrity of the plasma membrane will result in cell positive for Annexin V. The binding of Annexin V to phosphatidylserine can be affected in adherent cells, which are usally detached from plastic dishes by enzymatic treatment, although their membrane integrity is not altered.....

The link where i read it---

http://www.cyto.purdue.edu/flowcyt/research/cytotech/apopto/data/chap16.htm

The more we find out about something in first stages.......the more is the confusion hehe....but this is the way one gets crystal clear once minor hindrances are cleared so still positive on it.

What is your idea about annexin in adherent cells- u performed them in adherent ones or suspension type cells earlier.......

@Sanjaya: You can use BrdU labeling to check both apoptosis & necrosis.