Hi,
For a multicolored flow Cytometry experiment, we need to perform compensation of spectral overlaps (spill-over) due to usage of fluorochromes whose emission spectrum overlaps with each other. For doing so, companies provide compensation beads, which are uniform in size and give outputs as distinct positive signals for dim as well as abundant markers. Whereas, setting compensation by experimental cell may not be equally bright or distinct due to differential level of their expression. But with experimental cells, we get a real picture of spectral overlap and compensation will be done ONLY to the required extent. Moreover, here morphological changes expected to occur due to processing will also be reflected. But some time some markers are so dimly expressed that compensation cannot be performed by them. Thus is it better to take samples for compensation of flourochromes which are available with cellular markers present in abundance and we can use COMPENSATION BEAD for those fluorochromes vailable ONLY with dim/intracellular markers. means a combination of compensation beads and experimental sample cells can be used for setting compensation.
Thanks
For my multi-colour panel, I used cells to compensate Live-Dead dye and as unstained control while I used the beads for the rest of the panel. I can say using mix of beads and cells would be fine as long as you check that the voltages for all the flurochromes are suitable for both and never change them after the compensation has take place.
HI
What Mohammad said is right. The compensation is determined by the spectral properties of fluorochrome unless the marker assigned to the fluorochrome show high expression.
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