Testing New Fluorophores

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Presentation transcript:

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

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

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):

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

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

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