1. Testing New Fluorophores
Daniel Nissenbaum
Introduce yourself
I've been testing new fluorophores to use in addition to lab’s GFP and tdTomato over the past two weeks

2. Goals and Methods Test visibility of each Fluorophore
New Plasmids
eGFP
TagBFP
AmCyan
E2Crimson
HcRed
TurboFP650
Test visibility of each Fluorophore
Transfect 293T cells with the plasmids
View cells under fluorescence microscope
Test usability of each Fluorophore
Run transfected cells through LSRII
Look for bleed-through
Others
eGFP-DR
ZsGreen
ZsYellow
First Goal: test if fluorophores worked
Mention that all fluorophores worked
Second: Are they useable

3. Fluorescence Microscope Images (10x)
eGFP
TagBFP
AmCyan
E2Crimson
HcRed
TurboFP650
Red (TRITC):
Green (FITC):
eGFP shows up nicely under FITC filter
TagBFP is not very bright under DAPI filter but it is very clearly visible.
TagBFP as showed a very little bit of bleed-through under the FITC filter but only at very long exposures
AmCyan was not a very good fluorophore. Few cells fluoresced
E2Crimson shows clear bleed-through onto FITC filter.
HcRed also had few transfected cells.
TurboFP650 was very brigh.
Blue (DAPI):

4. LSRII (FACS) TagBFP AmCyan E2Crimson HcRed TurboFP650 eGFP mCherry
Alexa Fluor 488
TagBFP exhibits little bleeding into other channels
E2Crimson is all over the place and is remarkably bright
Turbo shows up very brightly in mCherry channel
Blue channel only picks up TagBFP (and a little E2Crimson, which is very surprising)
Alexa Fluor 350

5. More FACS Data No Plasmid TagBFP tdTomato TurboFP650 GFP mCherry
Alexa Fluor 488
Things to point out:
This GFP is the lab’s GFP
GFP is specific to the Alexa Fluor channel, with little bleed-through to the other two and very bright on the green channel
TagBFP is also has little bleed-through and works very well. It’s also distinguishable through the microscope and through FACS
tdTomato vs. TurboFP650
tdTomato, has been known to work but in this data, not very many cells were fluorescent, which is a bit of a surprise.
TurboFP650 on the other hand, shows up with much more brightness and many more cells, even though the bleed-through is a bit greater.
The bleed-through may be able to be compensated for
Alexa Fluor 350

6. Conclusions TagBFP works very well
Visibly distinguishable
Shows little bleed-through in FACS data
TurboFP650 may serve as a replacement for tdTomato, provided its bleed-through can be compensated for
Toxicity has not been addressed
For now, the best fluorophores are GFP, tdTomato, and TagBFP