The flow cytometer that I'm using just have the option of width and area for the FL2 detector (yelow/orange fluorescence), but I'm just using the green and the red detectors (FL1 and FL3). For those two, there is just option for height, what should I do in this case? how can I discriminate doublets?
Thank you
I usually gate doublets with the help of FSC-A and FCS-H (or FSC-W). However, if you are using an older Flow Cytometer, like for example BD's FACS Calibur, there is only the FSC-H option. This means, that you can't detect doublets at all. The same goes for the biexponential/logicle display.
This is a good paper explaining some difficulties regarding doublets discrimination:
Wersto, R. P., Chrest, F. J., Leary, J. F., Morris, C., Stetler‐Stevenson, M., & Gabrielson, E. (2001). Doublet discrimination in DNA cell‐cycle analysis. Cytometry, 46(5), 296-306.
I hope you will find it useful.
Actually, with the CellQuest software you can choose whether you want to analyze width and area in FL1 or FL3 channels.
When you begin your acquisition, click Cytometer and then Detector/Amps window. Click DDM Param, you will this option in the lower right corner of Detector/Amps window (the default option is FL2) and you will see FL1 and FL3, then you will be able to analyze area vs width in FL1 or FL3 channels.
This is option specially useful if you are staining with 7-AAD for cell cycle analysis. However, the analysis is made on lineal scale. Hope this helps.
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