I'm using panc02 cells, mouse pancreatic cancer cell line.Beforehand, I had some issues without doing any fixation, so now I’m trying with fixed cells.
IF you are measuring APOPTOSIS then it defeats the purpose of using the dual dyes.
* Acridine orange will stain both live and fixed cells. In live cells fluorescence is weaker than fixed cells because it is primarily accumulated by lysosomes in live cells, so staining of nucleus is weak, since under low concentrations it does not reach nuclei at high enough amounts. In fixed cells AO will stain ALL nuclei bright green (double-stranded) and nucleoli (RNA containing, single stranded) orange/orange-red. This brightness is strong.
** Ethidium bromide does not stain intact live cells. Only stains live cells with compromized membranes like dead or apoptotic cells. This is a bright orange-red fluorescence. In fixed cells the brightness and color remain somewhat the same and ALL the nuclei will still be orange.
So in live cells, living non-compromised cells will be a diffuse green (from AO) without very clear nuclei (depending on the concentration used), and apoptotic cells will SELECTIVELY be orange from EtBr.
IN FIXED CELLS, ALL CELLS will be equally green+orange (some shade of yellow).......and no way to distinguish them. So, no, there is no point in using fixed cells if you are trying to measure apoptosis. Indeed, there is really no point to using a combination of dyes on fixed cells, since they will all stain all nuclei.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction