Hi :D
I have done check up autophagosomal activity in RAW264.7 cell.
I was suppose to do my experiment as work flow under described.
1. drug treatment.
2. lysotracker treatment (DNS-99 RED)
3. wash 3times (for 3min each time)
4. fixation using 2% formalin
5. wash 3 times (for 3min each time)
6. permeabilization by 0.1% triton-X for 5min
7. wash 3 times ( for 3min each time)
8. treatment of LC3b primary Ab (O/N)
9. wash 3 time
10. secondary Ab (RT for 1hr)
11. wash 3 times and DAPI staining
12. wash 3times and imaging using confocal microscope.
When I used HT-29, human colorectal cancer cell line, It confirmed lysotracker and LC3b expression. But in RAW264.7 , mice macrophage, it didn't work.
What's the problem in my protocol?
Please help me to solve my trouble.
Hi D, The problem is the Invitrogen LysoTrackers are not fixable so the fluorescence signal is lost. I would suggest you use the better autophagosome specific dye, CYTO-ID from Enzo Life Sciences which is fixable Regards Gary
Thanks for your sincerely response, Gary.
Actually, my colleague have well done checking up autophagosomal activity by using LysoTracker DNS-99 on HT-29, a colorectal cancer cell line. So I did my exp. by his protocol. however, I didn't show data like my colleague.
If you know the different, let me understand this problem.
is there problems just dye? or other things?
- Badges
- Science topic