I am trying to do flow cytometric analysis of exosomes for my thesis work. The flow cytometry device I use can only distinguish 3 colors. I think of using each color for a different purpose. I will use FITC to verify exosome existence, PE-Cy7 to determine the exosome origin, and PE to determine the role of the exosome. I will use TSG101 conjugated with FITC to confirm the presence of exosomes. In addition to TSG101, I would like to add FITC-conjugated syntenin-1 antibody into the same antibody cocktail to show more exosomes. My goal is just to verify exosome existence, see FITC radiation. My goal is not to specifically show the presence of TSG101 or syntenin-1. So I think it wouldn't be okay to use two separate antibodies labeled with the same fluorophore. But I wanted to ask a specialist. Do you think that using two different antibodies labeled with the same fluorophore at the same time would create a problem? What kind of trouble does it create?
Hi Emel Karakaya,
As your purpose is to see TSG101+ and Syntenin1+ double-positive population to verify exosomes, it is perfectly fine to combine those two markers in the same channel (in your case FITC). Especially, given the fact that you have a limitation with respect to the instrument configuration (available detectors on your instrument).
In Flow Cytometry, it is a common practice to combine several markers in the same channel when appropriate. Such channel is referred as either "Dump Channel" or "Inclusion Channel" depending on the objective. In your case, this could be referred to as "Inclusion Channel" as you are interested in including double-positive population towards your analysis/sorting.
Depending on the level of TSG101 and Syntenin-1 on exosomes, you might see two or more distinct populations in terms of the fluoresence intensity of FITC (such as FITC-lo and FITC-hi populations). This is perfectly fine.
You can prepare single color controls to measure marker-specific fluorescence intensity in FITC channel (exclusive to TSG101 and Syntenin1 signal). This might further help you correlate FITC-lo and FITC-hi populations with the TSG101 and Syntenin1 markers (if applicable). Please make sure to include an unstained control, as well!
Hope this helps! Happy Flowing!!
P.S: I would be interested to see how it shows up. Please post an image here once you try it, if that's OK? :)
Dear Emel Karakaya,
I do agree with Javid. you may use single fluorochrome for two or more different antibodies to utilize as inclusion of dump channel. Your's will be the inclusion channel (I uses as dump channel to test double negative T cells).
And to distinguish between two different population of TSG101 and Syntenin-1 positiveyou should run single color tubes for both the antibodies along with unstained control. it will help you set the MFI of two different antibodies, which will further help to distinguish the two exosome populations (if there is any difference in MFI of single color).
Go ahead with the experiment and please post the image of your results here so we may also see it.
Hope this is helpful.
Dear Javid Mohammed and Jitendra Kumar Shandilya ..... Thank you both very much. Your answer encouraged me. When I perform the flow analysis, I will definitely share the images I obtained here.
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