hiii saswat, you can use neutral red uptake assay i think it works

you can measure LDH activity, leaky membranes releases the LDH into culture supernatent

Eithidium homodimer is not a cell permeable fluorescent dye, and it enters the cell when membrane integrity is compromised. It is a component of Live/dead assay kit. It should help.

Thanks Both of you for your valuable suggestions. But Here, peptide itself induces apoptotic death, most probably by inhibiting cellular cytoskeleton. Again, it might be causing catastropic death. Thus I need to distinguish whether the cell death is because of membrane lysis or the pathway we already observed (by cytoskeleton). The methods suggested above might not be able to discriminate the membrane lysis, necrosis or catastrope or late phase of apoptosis in which similar results will be observed. E.g may be the peptide is not acting like detergent and does not affect the membrane i.e negative in membrane lysis, as it is doing apoptotic death or necrotic for which we may got positive results in both the suggested experiments. Can you please suggest any specific experiments so that we can be able to discriminate both the possibilities..

You  may add a fluorescent caspase substrate of another color (green) to detect apoptosis in addition to Ethidium homodimer (red) to detect necrosis/secondary necrosis

Hi Saswat,

We usually use trypan blue staining to see if membrane is intact or not (please see https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection OR http://www.sciencedirect.com/science/article/pii/S0042682214000117). It is challenging to do simple color stains and have definitive proof if it is primary or secondary necrosis. Saswat, you may have to do a combination of apoptosis markers as Goodwin pointed (you can use active caspase 3 as in the paper above) with dyes that will not penetrate you plasma membrane like trypan, but i don't know if these two is compatible since immunostaining has a permeabilization part that can let big dyes get through the membrane into the cell. Maybe you have to do them separately with your peptide. Best wishes!

-ED 

Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...

try propidium iodide, it should only stain cells which have lost their membrane seletivity thus indicating necrosis. 

Maybe time-lapse analysis would help if you can observe blebbing.

https://www.researchgate.net/publication/41893623_Time-Lapse_Analysis_of_Cell_Death_in_Mammalian_and_Fungal_Cells

Article Time-Lapse Analysis of Cell Death in Mammalian and Fungal Cells

to detect membrane damages I will first use trypan blue stain together with LDH release to test rapid membrane alterations. I will also study the time course of the lethal effect.... if it occurs within minutes or less than 1-2 hours, it is probably related to either detergent effect or to membrane lysis.... if it occurs after more than 12-24 h, it will probably relate to apoptosis... you may thus test specific markers of apoptosis such as DAPI to observe apoptotic bodies but may also test whether or not toxicity could be blocked by inhibitors of either protein or RNA synthesis...

If you have in hands analogs of your peptide you may search if small modifications of side chain charge or the presence of lipophilic/hydrophilic chains may alter toxicity in order to determine a possible structure-activity relationship and the eventual presence of specific receptors onto the membrane of your cells.

Thank you all for  sugestions. 

Thank you Dominique,

for valuable suggestions. I am willing to perform the experiment. For the time lapse experiment I need any previous reference available. Can you please suggest any.   

Hi, you may have a look on the paper by Vimard et al. Toxicity induced by cumene hydroperoxide in PC 12 cells: protective role of thiol donors. J Biochem Mol Toxicol 25, 2011, 205-215.

Good luck

Dominique

Dear Dominique,

Can you suggest any reference for LDH assay time dependent manner.

With regards,

Saswat

If you use a cell culture flask with a surface of 75 square centimeters and a culture volume of 10-12 ml, you may take  100 µl aliquots sample at various intervals following cell treatment to measure LDH release. If you use multiwells plates, treat your wells at the same moment and then measure LDH release in one of the wells, assuming that all the wells behave in a similar way.

Good luck

Dominique