GFP+ Transf. HEK293 GFP- in fluorescence microscopy? - (Jun/14/2015 )
I'm confused and therefore write here with the hope that someone might provide useful input. Maybe it's too obvious and I just happen to miss the forest for the trees.
I've stably transfected HEK293 cells using HD Fugene. Once I transfected them with a EGFP-only-vector, once I used a vector with my gene of interest before EGFP. Afterwards I selected positive cell clones using G418/Geneticin (mock- and untransfected HEK293 cells died while doing so).
From flow cytometry I can clearly see that all of the colonies picked (doesn't matter which construct) are EGFP-Positive, some more, some less. Yet when using fluoresence microscopy only the colonies transfected with the GOI-EGFP-vector show GFP-fluorescence, while none of the EGFP-transfected HEK293 cells seem to exhibit GFP-fluorescence. If that matters: Cells are put on slides using cytospin centrifugation for fluorescence microscopy.
Does that sound familiar to anyone or has anyone else encountered this problem before?
Great thanks in advance!
I've did some research and even though I'm not entirely happy with the outcome, I wanted to let anyone know what I've found so far. At the end of my posting, you'll find the paper/resources I'm referring to.
After my cells are cytospined, I fixed them for 5 minutes in absolute ethanol at RT. It seems this is not a really beneficial treatment for conserving GFP fluorescence. There are resources that state GFP fluorescence will be lost or altered after fixation in alcohol, such as ethanol or methanol. Either this is due to denaturation or due to perforation of the cell membrane, so that GFP will diffuse out of the cell. On the other hand, there is one answers, which Nybo collected, that a GFP-tagged protein shows indeed fluorescence.
So, personally I assume that fusion of GFP with my protein of interest somehow prevents (or diminishes) loss of GFP fluorescence, either due to increased size (so it does not diffuse out of the cell anymore) or that denaturation than occurs in a way, that GFP fluorescence is (perhaps to a lesser extent and/or only partially) conserved.
As always, I'm happy about input.
Brock et al. (1999). Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry. 35(4), 353-362.
Nybo, 2012 GFP imaging in fixed cells. Biotechniques. 52(6), 359-360.
Kalejta et al., 1997 Use of a membrane-localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry. Cytometry. 29(4), 286-291.
Kiernan J.A. (2006). Fluorescence of GFP in sections of fixed tissue. Biotech Histochem. 81(4-6), 159.
There is also a link of researchgate, where this topic is discussed: http://www.researchgate.net/post/GFP_fluorescence_after_fixation10