It's my first experiment in flow cytometry. I'm attempting to exclude non-single cells. I'm wondering why it's not common practice to simply discriminate using pulse width (Time of Flight)? What important events could I be excluding from my data?
I use a cell line, and not blood, so my samples should be homogeneous in size.
Please see the attached image.
You are probably referring to using the width of a signal pulse and that is in fact being used as well. For some reason, most people are plotting Area vs Height, which would give you this nice diagonal in your upper plot only if the Area Scaling Factors are set up correctly. If you are using cell lines, you probably will not be able to see this.
You can very well use Area vs width, which will then display your singlets as a straight line. However, none of these methods are perfect for excluding all doublets, and you should always check your particular cells by backgating to see whether you are excluding any relevant events.
Thanks Florian, Ive learned solely on a machine that uses terminology like TOF and Integral, and forget to use what most other people use such as Width and Area.
I came across another question here : https://www.researchgate.net/post/Purpose_of_signal_width_w_in_cell_cytometry2
And some people such as Jaka Lavrencak say they use Width to discriminate single cells.
To answer your question directly, it's not common practice because conventional flow cytometers (an LSR-II, for example) don't commonly record pulse width. Are you doing CyTOF?
No, not cyTOF, just using the Gallios Flow, doing cell viability annexin/PI on some drugs.
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