We just incorporated a Northern Lights Spectral Flow Cytometer, and we want to analyze glucose incorporation in T lymphocytes using the 2-NBGD probe. The thing is that we cannot find the spectral fingerprint (ribbon plot) from 2-NBDG. Anyone that could help us with this? And of course any information / advice you can give us will be deeply appreciated!
Hi Maria,
I have never worked with 2-NBDG, but it seems it is excited by the blue laser (488nm) and detected in the FITC channel on a conventional flow cytometer. I don´t know the laser configuration of your Northern Light flow cytometer, but I am guessing that most likely, you have a blue laser. If that is the case, you can measure 2-NBDG. It will probably be similar to FITC/SparkBlue 550/ AF532, PE(?) (max. emission at 550nm, according to google), so keep that in mind during panel design.
For unmixing, I would recommend to create a single stain control with the probe and using unstained cells (as you would anyways) as a negative control. that will give you the spectral fingerprint for the probe in the unmixing window. and may give you a good idea on how to implement it in future panels.
Make sure the single stain "expresses" the target for the probe and make sure the unstained control is stained exactly the same way as your single stain.
If you want to compare it to other fluorochromes directly, I would just import (or make fresh) other single stains of similar fluorochromes (FITC, SparkBlue550, AF532, maybe even PE). When you are in the unmixing window, you can see the spectra (click "next" after gating your single stains). Additionally, you can calculate the similarity score matrix in that window and export it. It is not as easy as using the spectra viewer from the Cytek Website, but in the end, you´ll get the same results. Perhaps you could ask them to upload the 2-NBDG spectrum to their database, but that would probably be too late for you :)
Hope this helps!
Best
Johannes
Hi! Thaks a lot for your answer Johannes and Lyudmila!
I will keep in mind your valuable advice. Yes, we have a NL with three lasers, a B-R-V configuration, therefore the excitation/emission is exactly the way you described....
Ee already ask to our FC specialist for the 2-NBDG spectrum, I hope we will have it soon.
Best,
Florencia
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