Hi,
I want to determine the mechanism of programmed cell death in cancer cells due to our compound. I want to study necrosis but unable to find any kit. Trypan blue staining is recommended for studying necrosis. I would like to know the protocol for the same.
For apoptosis, I am using Annexin V FITC but not finding anything for necrosis.
Ethidium homodimer of Live or Dead assay (can be bought separately). See my paper on secondary necrosis
https://www.researchgate.net/publication/292615481_Mitochondrial_oligomers_boost_glycolysis_in_cancer_stem_cells_to_facilitate_blebbishield-mediated_transformation_after_apoptosis?_iepl%5BviewId%5D=lW4baka26CadAZ7XPYALMTGHDZpt0F3A4uoI&_iepl%5Bcontexts%5D%5B0%5D=prfhpi&_iepl%5Bdata%5D%5BstandardItemCount%5D=4&_iepl%5Bdata%5D%5BuserSelectedItemCount%5D=5&_iepl%5Bdata%5D%5BtopHighlightCount%5D=1&_iepl%5Bdata%5D%5BstandardItemIndex%5D=3&_iepl%5Bdata%5D%5BstandardItem3of4%5D=1&_iepl%5BtargetEntityId%5D=PB%3A292615481&_iepl%5BinteractionType%5D=publicationTitle
Article Mitochondrial oligomers boost glycolysis in cancer stem cell...
It depends if your cells are in suspension or adherent. If they are in suspension it is relatively easy to perform. I mixed in an assay tube 50 µl of commercially available trypan blue solution (sigma) with 50 µl of cell suspension and 200 µl of DMEM (without phenol red). Mixed gently and put an aliquot of the suspension on a Malassez Chamber. Then, wait 1-2 minutes and count your blue and white cells under a light microsscope.
If your cells are adherent, you have first to put them into suspension using a scrapper (so called rubber policemant) or a gentle treatment with trypsin.
Another way of measuring cell death is to use lactate dehydrogenase release in your culture medium after drug treatment. You may use an old but performant technique described by Wroblewski and Ladue: " Lactic dehydrogenase activity in blood" Proc Soc Exp Biol Med 90, 1955, 210-213
We used a commercial trypan blue at 0.5 %. We do prepare 0.2 to 1 million cells in 50µl in HBSS to have enought cells in the chamber. Addition of less than 50 µl of Trypan at 0.1% (diluted in HBBS). Thomas chamber is also OK.
For the lactate dehydrogenase, be aware that what you measure can be destroyed during long time (several days) of incubation and sometimes by the drugs. Impossible to perform in our case.
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