I am a novice in using the flowcytometer and currently I am using single stain, 7-AAD to differentiate the live vs dead cells of one cell line. My questions are:
1. How to properly gate without being bias?
2. Do we gate at the FSC vs 7-AAD or will it be better to gate using histogram? (comparing treated vs vehicle and dead controls) Because I find it is somewhat difficult to know what is the limit/threshold if I gate in the FSC vs 7-AAD dot plot without being bias. Some reference gate at the SSC vs FSC dot plot and never mention how to determine the gate border (how big or close should the gate go beyond concentrated dots). There are also others using histogram to gate rather than the other dot plot graphs.
I will be running PI stain soon though to confirm apoptosis and stages of the cells are in. It is just I am curious on how to properly gate the dot plot graph.
You make it up. "Ohh this looks like it might be a distinct cloud" is a perfectly acceptable gate definition. There's a lot of poetic freedom in flow cytometry. And that's a problem. But no one has come up with a universally accepted formal system to define the gates. Because they like it that way.
At least with 7AAD you have some fairly defined clouds that you can encircle in a reasonably uncontroversial way. Once you're looking at immunology markers, where the clouds partially overlap, you can basically get your data to say whatever you want, based on how you place the gates. Be wary of that :)
Hi Reagan
1. The limit/treshold will depend on the instrument and the voltage you are using. The voltage can also interfere with the resolution (separation of both populations). If you are not discriminating both populations easily, one thing that can help you is to use a "positive control" for 7AAD, such as cells incubated for 20min at 55oC or 200uM H2O2. These cells will be mostly (if not all) dead, so 7AAD pos, while your untreated cells will be mostly 7AAD neg. This can help you set your gates properly.
2. Personaly I prefer to plot 7AAD vs "dump channel" but since you are only stainning 7AAD you can use 7AAD vs FSC. Besides that I find the "contour plot" easier to work with than dot plot. You should give it a try.
Hi John and Andre, thanks for your feedback on the queries. Because having 2 supervisors with different opinions on gating so it has been a headache to get around this, so far my gating are based on the controls. It is just flow results were not tally with viability assay; probably limited ability of 7AAD to differentiate live, apoptotic and dead cells effectively thus causing the difference in results between assays. I was trying to at least matched the viability results through gating but this would be manipulating data and being bias which I want to avoid and unethical. Then again 7AAD would not be able to tell the whole story yet until I run PI soon. Thanks again for the feedback.
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