Why do viable cells take up DAPI? - DAPI must only stain non-viable cells (ICC) (Sep/13/2008 )
Hi,
DAPI is supposed to stain non-viable cells, at least this is what I've been reading everywhere.
I just don't understand why my healthy cells also take up the stain !!!
My adviser says DAPI can enter cells after fixation. If so then what's the point of using it?
maybe I should forget ICC, and do live imaging on cells grown in flask without fixation !!!???
In many papers DAPI is used to identify whether there is DNA damage or to identify cells with abnormal DNA like those that exit mitosis abnormally and form cells with micronuclei. Moreover it can be used to identify mitotically arrested cells among normal cells for calculation of mitotic index. ofcourse this is done after a fixation step.
if you want a marker for cell viability I think you should use propidium Iodide (fluorescent ) or trypan blue, where both can added to trypsinized cells after washing with PBS without fixation.
I haven't used DAPI but Wiki says DAPI stains both live and dead cells.
DAPI passes through live and dead cell membranes quite easily, however, fixed cells are permeabilised and so the uptake of the stain is much better. DAPI is usually used for detecting the nucleus in IF in co-localisation studies and such like.
If you want to distinguish live/dead try using something like MTT which works on mitochondrial activity
yup, that's what I'm seeing in my experiments. both live and dead take up DAPI.
I'm planning to run a test with cells grown in flasks, without fixation/permeabilization. I'm just gonna remove media, wash with PBS and then add DAPI for 5 min. then immediately observe under inverted fluorescence microscope (before they lift up).
will let you know about the result Bungalow...thanks.
yup, that's what I'm seeing in my experiments. both live and dead take up DAPI.
I'm planning to run a test with cells grown in flasks, without fixation/permeabilization. I'm just gonna remove media, wash with PBS and then add DAPI for 5 min. then immediately observe under inverted fluorescence microscope (before they lift up).
will let you know about the result Bungalow...thanks.
Will be looking forward to hear the conclusion of this experiment. I am not sure what is the objective though.If you want something that does not penetrate live cell membrane, how about PI or 7AAD or MTT ? or TUNEL or Annexin V for apoptotic cells.
no, the objective is something else. PI can also be useful for our case. but TUNEL and Anexin are completely a different story.
How was DAPI on the flask cells?
And what about 7AAD