Please take a look at the attached files:
I am about to study 2 markers which appear in FL1 and FL2;
In the unstain tube, I see 2 distinct populations one of which has weaker auto-fluorescence signals (pop 1) and the second (pop 2) with stronger auto-fluorescence signals which appear in front of the other population!
In the first image:
On the left is the unstain tube on which pop 1 is gated and on the right is the corresponding SSC/FSC plot.
In the second image:
On the left is the same unstain tube on which pop 2 is gated and on the right is the corresponding SSC/FSC plot.
As you see, the two populations appear at different areas in the SSC/FSC plots confirming that they are different cells with different size and granularities.
My 1st question is why do these 2 populations appear in different areas in my unstain (FL1/FL2)?! Is it because they have different sizes and granularities as shown in the SSC/FSC plot?!
2nd: Since these 2 distinct populations exist in my unstain tube, where should I put the quadrant to identify the cells positive for the markers which appear in FL1 and FL2? As you see in images 3 and 4, I would lose part of the population at both of the circumstances shown for the quadrant in images 3 and 4!!
How can I deal with this?
I would highly appreciate if anyone could help me out!
Thank you in advance
Q1: The autofluorescence of different cell populations, as you suggest, is based on the size and complexity of the cells. Imagine your shining a flashlight beam on ballons in the dark. If your beam hits a large balloon you will see more light scattered back at you than from a small balloon. Autofluorescence affects all channels, not just forward- and side-light scatter.
Q2: I would use gates based on forward- and side-light scatter to distinguish cell populations and then compare your staining on these populations. Your sample looks like it could be blood, at least small lymphocytes and high-side scatter granulocytes. If you distinguish these populations by light scatter profiles then you will mostly separate the two populations you see in FL1 and FL2. If you need more separation, then you need to do additional stains to gate-out or gate-in subpopulations.
One way to deal with the issue is to make two separate gates based on the forward and side scatter plot as the first thing you do i.e set gate 1 as "FSC low SSC low" and gate 2 as "FSC hi SSC hi". Then set the FL1-FL2 quadrant gates separetly for the two different populations. In this way you will largely avoid false positives and false negatives each population.
Size and granularity (/inner complexity) often explains autofluorescence, but they are not always the reason - e.g. dead cells often have a higher background signal as well.
Joakim
Hi
Have you stained your cells with live/dead stain and also have you taken care of doublets in your data.
I would recommend this before making any assumption on these being two distinct populations.
Thank you for your detailed and helpful responses Dr. Dahlim and Dr. Muench. I will definitely address your comments
Dr. Amin Dar,
I have stained the cells with PI and most of them are alive.
I have also done all preparations to remove doublets.
Best
Dear Milad,
I am agree with Marcus and Joakim to first determine your cell population based on FSC-H and SSC-H, and then set FL-1 and FL-2 quadrant.
When you first plot FL-1 and FL-2, you are considering all of the cells which have different auto-fluorescence.
Also regarding doublets discrimination, you should include FSC-A vs FCS-H, FSC-W vs FSC-H or etc in the cytometer parameters when you are setting your experiment . You can find additional suitable information in this regard in the link below:
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=11&cad=rja&uact=8&ved=0ahUKEwi8vYqisfvXAhVGAZoKHeUDC0sQFghSMAo&url=https%3A%2F%2Fexpertcytometry.com%2F6-flow-cytometry-gating-tips-that-most-scientists-forget%2F&usg=AOvVaw3F712DPJd6WS2T6NYKNslC
Best
Dear Dr. Zare,
Thank you for your useful information.
Best,
Milad
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