My work aims to observe cell viability through AO/PI staining. Although I found numerous AO/PI staining protocols, but most of them analyzed the viability through microplate reader or cell counter (countess).
We're trying an approach of observing these cells under microscope with fluorescence setting. I plan to seed my cells into 96 well plates, and later stain them with AO/PI staining. However, I couldn't find any detailed protocol for this.
It seemed like AO/PI stain was diluted with PBS, prior staining - but this was for microplate & countess protocols.
Should I dilute my AO/PI stain? if yes, how much volume is needed? I'd also like to know if there is any incubation period before observing my stained cells under microscope.
Thank you!
Hello Luqman!
I think, you couldn't find this protocol, because both of AO and PI have similar luminescence spectrum as DAPI, Hoechst etc. and nobody can't differ they. You may stain cells AO/EB, we use DAPI-similar (340 — 380 / 435 — 485 nm) and 525 — 540 / 605 — 655 nm filters.
We use fixation by 4% formaldehyde in PBS for 30 min and staining by 100 mkg/l AO and 40 mkg/l Ethidium Bromide for 30 min.
Good luck!
AO/PI staining is a technique used to determine the viability of mammalian cells by staining them with acridine orange (AO) and propidium iodide (PI). The protocol for performing AO/PI staining on mammalian cells in 96 well plates is as follows:
AO/PI staining is a useful technique for determining the viability of mammalian cells in a 96 well plate format. It is important to follow the protocol carefully and use proper laboratory techniques to ensure accurate and reliable results.
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