Help with Tunel assay Fluorescein - (Apr/17/2008 )
Hi folks,
I am interested in examing apoptosis in my cells hence i've just started the Tunel assay Fluorescein from Roche.
I followed every single step in the protocol, however when i ran my samples on the calibur, i couldn't see the positive population of cells that were apoptotic, even in my treated cells and positive control. All i could see was one beautiful negative peak. Hence i was wondering if any of you kind folks have got a protocol that works. Another question of interest is, do you guys just gate the main population of cells in the forward scatter and side scatter plot and plot that into the histogram with FL1 to analyse apoptosis?
Many thanks
wei
-weii02-
QUOTE (weii02 @ Apr 18 2008, 02:42 PM)
Hi folks,
I am interested in examing apoptosis in my cells hence i've just started the Tunel assay Fluorescein from Roche.
I followed every single step in the protocol, however when i ran my samples on the calibur, i couldn't see the positive population of cells that were apoptotic, even in my treated cells and positive control. All i could see was one beautiful negative peak. Hence i was wondering if any of you kind folks have got a protocol that works. Another question of interest is, do you guys just gate the main population of cells in the forward scatter and side scatter plot and plot that into the histogram with FL1 to analyse apoptosis?
Many thanks
wei
I am interested in examing apoptosis in my cells hence i've just started the Tunel assay Fluorescein from Roche.
I followed every single step in the protocol, however when i ran my samples on the calibur, i couldn't see the positive population of cells that were apoptotic, even in my treated cells and positive control. All i could see was one beautiful negative peak. Hence i was wondering if any of you kind folks have got a protocol that works. Another question of interest is, do you guys just gate the main population of cells in the forward scatter and side scatter plot and plot that into the histogram with FL1 to analyse apoptosis?
Many thanks
wei
Hello wei,
I'm not familiar with Roche's protocol, but I've found that some cells require a longer incubation time with the tdt enzyme than the 1 hr suggested in most protocols. My cells require at least 3 hrs at 37C in the dark with resuspension every half hour for the TUNEL positive peak to appear. As for gating, since my TUNEL kit came with a PI/RNase counterstain, I just gate using the FL2-A/FL2-H thingamajig.
good luck!
-subclavian-