2 replies to this topic

[aks]

Hi,
I am growing MCF-7 cell line. I have been trying to stain dead cells using ethidium bromide and I havnt been very successful. I found the protocol online but it doesnt seem to work. if there is any other method with which I can stain the cells please let me know. Thanks in advance.

Protocol
1. grow the cells on a cover slip coated with Ploy-L-Lysine
2. put one drop of ethidium bromide (5ug/ml) on the cells and incubate for 10 mins
3. rinse the cover slip with PBS
4. expose to UV light and the dead cells will be red in color.


and what wave length is ideal to excite Ethidium Bromide?

[doxorubicin]

It sounds like you can just use trypan blue and a hemocytometer, as in normal cell counting.
If you want to use a flow cytometer, consider switching to propidium iodide, and this protocol (for either EtBr or PI) looks pretty good:

http://www.immun.pit...l... dyes I.doc

[zodiac1505]

aks, on 15 June 2010 - 10:08 AM, said:

Hi,
I am growing MCF-7 cell line. I have been trying to stain dead cells using ethidium bromide and I havnt been very successful. I found the protocol online but it doesnt seem to work. if there is any other method with which I can stain the cells please let me know. Thanks in advance.

Protocol
1. grow the cells on a cover slip coated with Ploy-L-Lysine
2. put one drop of ethidium bromide (5ug/ml) on the cells and incubate for 10 mins
3. rinse the cover slip with PBS
4. expose to UV light and the dead cells will be red in color.


and what wave length is ideal to excite Ethidium Bromide?

If you  want to look for dead cells by FACS, you could use annexin (early apoptosis marker) or PI (late apoptosis marker). If you need protocols on how to do it, just let me know