We have a problem with bone marrow-derived dendritic cells. Has anyone experienced highly autofluorescent bone marrow-derived DCs and how have they dealt with this problem?
We sometimes experience similar problem. You did not mention which method you are using, I hope my answers are still helpful.
Flow cytometry : Try to use APC or PE with tandem labeled antibodies instead of FITC and PE. If you have no choice, select the most abundant marker for green channel and use unstained control to detect the auto-fluorescence
In vivo DC migration : There we pulse DCs with APC/Alexa650 labeled ovalbumin instead of FITC/Alexa488 labeled one.
LSM/confocal imaging : Same here, try to use channels with higher wavelengths as well as UV.
We sometimes experience similar problem. You did not mention which method you are using, I hope my answers are still helpful.
Flow cytometry : Try to use APC or PE with tandem labeled antibodies instead of FITC and PE. If you have no choice, select the most abundant marker for green channel and use unstained control to detect the auto-fluorescence
In vivo DC migration : There we pulse DCs with APC/Alexa650 labeled ovalbumin instead of FITC/Alexa488 labeled one.
LSM/confocal imaging : Same here, try to use channels with higher wavelengths as well as UV.
Yes, we were actually discussing your first option this morning when your answer came through. We will try the third option for our confocal imaging.
Thanks a lot.
You can try to shorten your protocol of generation of BM-DCs. The time spent in culture affects their autofluorescence intensity. Another option is to obtain DCs from purified monocytes from the BM (by positive selection with anti-CD115). This protocol generates DCs in 24h in the presence of GM-CSF and the DCs obtained are not autofluorescent. Hope this helps.
@Beatriz,
i will see if we can get hold of CD115 and try out shortening the production time.
Thanks
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