Hello, I got this result from my experiment. i wanted to learn there is apoptosis or not in my treated cell lines. My protocol is
Firsly, i added 4 ul of acridine orange to my treated cells in t25 before harvested.I did not add to non treated and vehicle groups.
then 15 min. incubation.
ı took medium to falcon tube and i harvested with tripsin after that i added to this. falcon tube again.
aftwr 5000 rpm 5 min centrifugation,i washed with pbs 2 times. and added 300 ul pbs to each one then i read with flow cytometer. (cytoflex)
But this results shows nt and vehicle groups are dead cells. Actually i really want to learn how can i analyze this result.Also, after that i will use annexin pı to detect apoptosis but fiisly ı wanted to use acridine orange protocol to detect apoptosis. is it true process or hıw can i detect apoptosis with a.o..
And what is the means of this results?
This flow cytometer protocol is ok?
i really want to know how can i use and analyze this flow cytometer results.
Dear Berna Özdem ,
Acridine Orange dye is know to give green fluorescence when bind to DNA and Gives red color when permeate to lysosomes.
SO, when you open a bi-axis (FITC/PE) dot plot on gated cells, you will get FITC only cells (Which are basically Dead cells ), the PE only cells are live cells (membrane is intact) and orange signal give you the count of apoptotic cells.
Dear Berna Özdem ,
Acridine Orange dye is know to give green fluorescence when bind to DNA and Gives red color when permeate to lysosomes.
SO, when you open a bi-axis (FITC/PE) dot plot on gated cells, you will get FITC only cells (Which are basically Dead cells ), the PE only cells are live cells (membrane is intact) and orange signal give you the count of apoptotic cells.
Hi Berna
You need to process all the cell pool with same way for best analysis for your study. By this test, if you find more apoptotic cells as compared to nt and vehicle treated cell pool from your drug, your drug could be potential anticancer drug.
thank you so much to all.İ will also add acridine orange to nt and vehicle.
But, according to this result ll is live cells lr pro apoptotic. cells but in tihs result nt live cells also looks dead apoptotic cells? how can i fix that? Actually i want to learn how can i analyze and use this flow cytometer. Do if you have any articles or some books i will be appreciate. thank you so much for your interest ☺
- Badges
- Science topic
- Similar topics
- Psychology
- Mental Processes
- Learning