I use transcription factor buffer set from BD. I have not changed anything but I started to get two peaks in my Aiolos staining in B cells. I got two peaks for another transcription factor which used to be one peak as well. any idea/comments/experience ? Thank you so much.
Dear Yumi Nakayama,
have you checked the flight time of your cells in the FCM data? It may be that you are getting cell aggregates, which can be identified through their fligt time. Aggregates will cause a second peak. Kind regards, Harrie Verhoeven.
Dear Harrie,
Thank you for the comment. could you tell me what "flight time" is in FCM data? I have never used flight time.
Sincerely,
Yumi
Dear Yumi,
Flight time is the width of the pulse (on some machines it is included in the data as pulse width) caused by the passage of particles under the laser beam. If two cells are clumping, you will get a wider pulse, in combination with a higher intensity. In this way you can discriminate between G2 cell phase cells and two G1 cells clumping together. Scatter signal could do this also, but less accurate. Pulse width is not available on all instruments. I used it on a Cyan FCM with very good discrimination between singles and aggregates. Kind regards, Harrie Verhoeven.
Dear Harrie,
Thank you for much for the followup and sorry for the late reply.
For that experiment, I did not have the width but I will include for the next to see the difference.
Sincerely,
Yumi
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