I want to use DAPI as a marker for dead cells but also want to fix these cells with 2% PFA.
As DAPI can enter permeabilized cells, I will do this before my permeabilization step.
However, would the fixation interfere with the use of DAPI as a dead cell marker?
Thank you in advance!
Hello Madelief Marsman
DAPI is somewhat less cell membrane permeant, although the stain will enter live cells when used at higher concentration (usually 10ug/mL) for live cell staining. You can use DAPI for fixed cell staining at 1ug/mL.
Best.
Hello Madelief,
you can use propidium iodid as a marker for live/dead cells... You have to test the right concentration (about 2 µg/mL in your medium). You can incubate your cells for 1 min up to 5 min...Than you can fix them and stain with dapi (1:10; 5 min). All dead cells will be positive for propidium iodid, because propidium iodid can only enter the cells if the membrane is destroyed. Dapi will enter also an intact membrane and stain all cells. So you can build the ratio of dapi/propidium iodid....
best regards,
constanze
hi Madelief Marsman
You can not use DAPI for estimating the live/dead cells after fixation. If you want to estimate the dead cells then it has to be done before fixation. You can stain the cells with DAPI and then fix the cells with PFA for further analysis or process.
If you perform a DAPI staining after fixation, all the cells will take up DAPi and you will end up all dead result.
As advised, PI and annexin V are best for live dead assay.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Fixatives