Hi researchers,
I am currently working on some confocal cell uptake experiments of my graphene quantum dots (GQDs), which are naturally fluorescent. I am treating SHSy5Y cells with 100ug/ml of these GQDs, after which, I remove the media, wash the cells with PBS and fix with 3.7% PFA. I later mount with prolong antifade mounting medium.
The issue is, this procedure worked very well the very first time I did it, with bright fluorescence of my GQDs in the cells, even at a low laser intensity of 2%. Ever since, I had tried to reproduce it and I could only get a lot of noise (and I have to push the laser intensity to 100% to see anything, but problem is that even the untreated cells will fluorescence at such intensities, so it is not a true signal).
The only step I think is different is addition of serum when incubating my GQDs with the cells. The second time I tried incubating in serum free media, the cells died, hence I did not carry forward with that method. But i strongly feel that could be an issue that is causing interference with my GQD fluorescence.
A little background on the GQDs: These are fluorescent nanoparticles, but do not have a narrow emission range and hence can fluorescence when excited anywhere from 400 to 500nm. But upon entry into the cells, both the 488nm and 561nm laser lines could give me a signal. I have not been able to repeat this after the first round of experiment though.
Maybe I will also try incubating the GQDs for a shorter period, instead of 24 hours, but if anyone has had similar experience could you share with some tips?
Thank you!!!
Best regards,
Mathangi
Ashkan Zare Karizak ok thank you so much for the valuable feedback! I will take these into account during my next repeat.
Check your PFA fixation steps. specially if you have been using different batches of PFA. or if the current batch is old. pH is another issue.
Also yes, linear unmixing is possible in our confocals. So that’s is also a good idea if nothing else works.
Raghumoy Ghosh thank you for the input. Yes I have since checked the PFA steps as that could have been contributing to the autofluorescence. I tried a quenching with Sodium Borohydride step before fixing with freshly made PFA too, seems to decrease autofluorescence slightly but not so much. But now I have managed to adjust the channel settings in Zeiss such that I just use the same settings that give zero signal for untreated cells, for the nanoaprticle- treated cells too. Thank you!
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