How to compare the fluorescence staining signal in cells - (Sep/29/2008 )
Hi,everyone
I am doing the filipin staining of treated and untreated cells. I have taken a lot of pictures from these two samples separately using the same criteria(same magnification, same exposure time). Now I want to compare the staining degree between these two groups of cells, what measurement can I use and how?
Thanks alot!
syby
I am doing the filipin staining of treated and untreated cells. I have taken a lot of pictures from these two samples separately using the same criteria(same magnification, same exposure time). Now I want to compare the staining degree between these two groups of cells, what measurement can I use and how?
Thanks alot!
syby
Hi, I've done this experiment before. I used a confocal microscope and the software had the options to measure the intensity of fluorescnece coming from my cells. i did this experiment for DAPI and FITC.
It depends on the software that you are using. It'll give you some rates that you can compare your sample with your controls.
In the same software you are taking your pictures with, whatever it is (ours is Slidebook), you can do that; in Slidebook we create a mask on the image, define the area we want to know its intensity and then software gives you a whole bunch of statistics including mean intensity of the area median, min, max, ...
Thank you so much all your guys!
I really appreciate them all!
Have a fabulous day!
Best,
Syby58
Not explicitly stated, but you need to use a reference standard that will be the same across all the cells and compare your results based on that. DAPI is a good example of something that should work as a standard.
I am doing the filipin staining of treated and untreated cells. I have taken a lot of pictures from these two samples separately using the same criteria(same magnification, same exposure time). Now I want to compare the staining degree between these two groups of cells, what measurement can I use and how?
Thanks alot!
syby
Hi, I've done this experiment before. I used a confocal microscope and the software had the options to measure the intensity of fluorescnece coming from my cells. i did this experiment for DAPI and FITC.
It depends on the software that you are using. It'll give you some rates that you can compare your sample with your controls.
does it work well? which filipin you recommend? are there special needs for fixation and permeabilization (we use 4% pFA, and triton x-100/saponin)...
are you talking to me? because you replied to my post. we use filipin for another experiment, to block the endocytosis pathways in our cell lines, not for Immunofluorescence.
yes, we fix with Paraformaldehyde 3-4%, and then we permeabilize by a Triton x-100 buffer.