Dear Zuzana Parackova

You can check this interesting discussion on RG

https://www.researchgate.net/post/What_would_be_the_best_way_to_detect_pyroptosis_and_necroptosis_in_situ

And i hope this serves as a cost-effective article

Article Detection of Pyroptosis by Measuring Released Lactate Dehydr...

Best wishes

Hello

Pyroptosis is characterized by caspase-1 activation, GSDMD cleavage and membrane rupture. The measure of LDH release and GSDMD cleavage are good but you need to explore caspase-1 activation. You can do it by flow cytometry, using FLICA1 substrate (but it is a little bit expensive) or detect caspase-1 cleaved fragment by western blot. However the majority of caspase-1 is released in the supernantant so you may culture your cells in medium without serum (OIptiMEM) and precipitate it (see our paper Article HSP70 is a negative regulator of NLRP3 inflammasome activation

You can also try to detect caspase-1 in cell lysates (you need to load 120µg of protein).

The last possibility is to explore ASC speck formation by immunofluorescence. However you need antibody again.

Good luck in your experiments.

Hi Cedric,

thank you very much for suggestions. We are planning to screen several inflammasome inhibitors, therefore we need a really rapid and robust method. We use an automized high contest screening, so we need a protocol for microscopy basicaly. So maybe ASC speck are a possibility. Or maybe for the beginning a less specific detection and then try to study GSDMD cleavage. Still not sure :)

Bye

Z