How to determine FACS result - (Jan/12/2015 )
Hello everyone, I am new users to this website.
Recently I have done FACS to determine if my cells express specific antigen or not. I got results which confused me quite a lot, I don't know it is positive or not. I attached the results here. The green one presents negative control, and the red one is the sample. I could see a small peak shift however I am not sure whether my cell really expressed antigen of interests or not. I am looking forward to your advice.
Hello, Chen.
First of all, replicates can show the way.
But, looking this simple overlay, I don't see significantly positivity.
FL1 was the parameter chosen for this antigen. Maybe you should use a more brighter fluorescence - as PE - for get the most intense signal and eliminate all doubts (unless you have used a marker as the Horizon BB515, that is strong in green channel).
Please, talk more about your protocol and markers used. If you really have a difference, you can see better results changing protocol details to potencialize this signal.
I hope can help! =)
Welcome to the forum.
I'd also say there is no clear positive signal, the tiny shift is probably unspecific. I also agree that you could try to optimize your staining conditions - if your negative control doesn't express the antigen at all, there shouldn't be this second peak at all, which looks like some unspecific binding.
Thanks for the kind replies from Rochaff and Tabaluga.
Actually I am doing exosome FACS. First I over expressed one protein in cells and I found it might load onto exosome. I purified exosome from culture medium and found the protein fractionated to the same fraction as other exosome markers, such as CD63 in sucrose density gradient . So the next experiment I need to do is to make sure the protein of interest really load onto exosome.
Because exosome is too small to be detected by FACS, therefor I need to attach the exosome to antibody-coated beads before running FACS. I conjugated CD63 antibody to 4um latex beads, then I incubated exosome with CD63-latex beads, Because CD63 is membrane protein of exosome, therefor exosome can be precipitated by CD63 conjugated beads . I then immunostained the exosome- beads complex with first antibody for 1 hour , washed three times with PBS, and incubated with 100Xdilluted Alexa-488 for another hour, washed twice and analyzed by FACS. In the isotype control, I used Isotype IgG to conjugate the beads instead of CD63, incubated exosome with IgG-latex beads, stained with same first antibody and Alexa-488.
The above is how I did my experiment, any advice is welcome and I appreciate.
Hello, Chen.
Very nice and delicated experiment.
I ask you permission to give a brief opinion on your method.
In a FSC x SSC dot plot of this experiment, each event represents a single bead with many exosomes linked by anti-CD63. You'll need a little more information in this experiment, isn't? In this method you only will know that the exosomes are there, but there are something inside the exosomes? Perhaps a second color marker for the "intra exosome" protein will be necessary, and then you will looking for double positive populations. additionally, you could try to see this exosomes in a confocal microscopy, using two markers (a colocalization concept), for example: green dots + red dots = yellow dots. In this case, you don't need isolate the exosomes from the cells, but a higher resolution equipment should be necessary, considering the nanoscale of this structures (~100nm?).
Not sure if my suggestion makes sense or if I was clear. What do you think?
About the green marker, did you tried another dilutions?
Tnx for the reply, rochaff87.To observe exosome needs electronic microscope, and need experts who are familiar with ECM, that is why I turn to FACS. I think I better try another dilution of Alexa 488. Tnx for your advice.