I used unlabeled cells (first image) to set the gate and the second image is my positive control.
I am looking for percentage of green cells (FL-1, ''expressing'' cells in my experiment) that are also showing a high red signal (FL-4, ''Dylight 650).
From the images, it is obvious that the positive control is showing a higher FL-1 signal compared to the unlabeled cells, and in the ''Expressing'' gate there are many events (40%) that are also showing a high FL-4 signal (Dylight 650).
The problem happens when I am presenting the data as percentages - even though there are only a couple of events showing a FL-4 signal in the unlabeled control compared to tens of thousands of events in the positive control, those events are inside the Dylight 650 gate so the percentage of unlabeled cells in the FL-4 Dylight 650 gate is 100% of its parent population. Since those cells were not even labeled, this is probably just some kind of a background signal.
Is there a way to remove those events when I am, for example, making a table and doing statistical analysis? I know that I could just move the ''Expressing'' gate up so that there are 0 events there in the unlabeled sample, but I am trying to avoid that because in some of my other samples, most of the FL-1+FL-4 positive events are in the bottom part of that gate.
Hi Vanja,
there are a couple of ways to get there.
First, I would suggest to use "FL4-DyLight650" as your X axis parameter instead of FSC. This will allow you to create a gate on the FL1& FL4 double-positive cells, as those are the ones you are interested in, correct?
Another way of going about this is to analyze the pecentage of DyLight650+ cells out of your total cells/ population of interest. It won´t show up in the dot plot, but you´ll find that statistic in the Table editor>Edit>Add OR Edit column>Freq of... in FlowJo.
Two other points:
1. I would recommend to have a positive control "only FL-1 expressing". Meaning you stain only with your FL-1 Marker (or stimulate, if it is GFP or similar) but do not stain with DyLight650. If doing so, you could gate on that control and set your DyLight650 gate accordingly.
2. If some samples are very close to the DyLight650 background, consider trying to find a good negative control for DyLight 650. Unfortunately, completely omitting the DyLight 650 antibody/ molecule completely is not a great control, especially if your positive signal is very close to the unstained background. You can´t say for sure if you see a positive signal, or if you are detecting unspecific binding of your DyLight650 antibody/ readout-molecule. Here, titration of your molecule, the cells you are analyzing, washing, incubation temp/time etc. can play a huge role.
Hope this helps!
Rather than individually gating for FL1 and FL4, first put a gate on your cells depending on forward and side scatter, then open this 'Scatter gate' for FL1 (X-axis) and FL4 (Y-axis) and gate on double positive populations, either using quadrants or a polygon.
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