TITLE: Spectral Recording of Gene Expression History by Fluorescent Timer Protein
AUTHORS: Anna R. Tröscher, Barbara Werner, Nadia Kaouane, Wulf Haubensak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Monitoring spatio-temporal patterns of gene expression by fluorescent proteins requires longitudinal observation, which is often difficult to implement. Here, we fuse a fluorescent timer (FT) protein with an immediate early gene (IEG) promoter to track live gene expression in single cells. This results in a stimulus- and time-dependent spectral shift from blue to red for subsequent monitoring with fluorescence activated cell sorting (FACS) and live cell imaging. This spectral shift enables imputing the time point of activity </span><span style = "font-style:italic;">post-hoc </span><span>to dissociate early and late responders from a single snapshot in time. Thus, we provide a tool for tracking stimulus-driven IEG expression and demonstrate proof of concept exploiting promoter::FT fusions, adding new dimensions to experiments that require reconstructing spatio-temporal patterns of gene expression in cells, tissues or living organisms.</span></div></div>

[STEPS]
?. [Cell Culture and Transfection]
Grow HeLa cells overnight in standard incubator conditions (, 5 % CO2) on 6-well plate in standard medium (DMEM + 10 % FCS + 1 % Pen/Strep + 1 % L-Glutamine) to a confluency of about 80 %.
37.5 °C
?. [Cell Culture and Transfection]
Transfection with the TransIT-LT1 transfection reagent according to manufacturer: To , add TransIT-LT1 reagent.
[plasmid in Opti-MEM I reduced serum medium]
?. [Cell Culture and Transfection]
Incubate for  at .
37.5 Room temperature
?. [Cell Culture and Transfection]
Add reagent to cells for .
?. [Cell Culture and Transfection]
Remove medium containing transfection reagent, wash with PBS and add fresh standard medium.
?. There are alternative options for the following steps: Fluorescence-activated cell sorting (FACS) (alternative I) Life-cell imaging (LCI) (alternative II)
?. [FACS]
Leave cells undisturbed until induction (  are recommended).
?. [FACS]
Induction with TPA.
TPA is highly cancerogenic so wear respective protective gear!
TPA is very volatile: make sure you only add TPA to the wells, in which you want to induce the cells!
?. [FACS]
Baseline measurement: cells dislodged with trypsin and fluorescence and cell number determined by FACS.
?. [FACS]
Grow + and - TPA cells in separate 6-well plates to guarantee no TPA spills over into - TPA wells, as also the smallest amount of TPA induce IEG expression.
?. [FACS]
Incubate for  with .
[TPA diluted in standard medium]
?. [FACS]
Carefully remove all TPA and also remove medium of - TPA cells.
?. [FACS]
Wash with PBS.
?. [FACS]
Add fresh medium or add about  until cells are detached from the plate.
[trypsin]
?. [FACS]
Add  to the cells for deactivation.
[standard medium]
?. [FACS]
Pellet cells by centrifugation.
?. [FACS]
Resuspend in PBS.
?. [FACS]
Perform FACS measurements at the time points of your choice.
We recommend every two hours for the first 9h and again at 24h post-induction.
?. [FACS]
You can analyze FACS data with any standard FACS analysis program (e.g. FlowJo).
?. [Life-cell imaging]
Transfer cells into chambered cell culture slides  after transfection.
?. [Life-cell imaging]
Take cell growth into account when determining your cell density seeded onto the plates as they will stay on the culture slides for another .
?. [Life-cell imaging]
Let cells rest for  in the incubator on the cell culture slides.
?. [Life-cell imaging]
Induction with TPA.Add .
TPA is highly cancerogenic so wear respective protective gear!
TPA is very volatile: make sure you only add TPA to the wells, in which you want to induce the cells!
[TPA diluted in standard cell culture medium]
To avoid inducing – TPA cells with TPA, cover wells with parafilm while working with TPA. TPA is very volatile and the smallest amounts are enough to induce IEG expression.
?. [Life-cell imaging]
Incubate cells for  with TPA.
?. [Life-cell imaging]
Remove all medium (also from - TPA cells), wash cells with PBS and add fresh medium.
?. [Life-cell imaging]
Image cells with fluorescent microscope capable of life cell imaging over .
We recommend imaging every hour.
?. [Life-cell imaging]
You can now analyze the images with any software of choice (e.g. ImagePro Premier, ImageJ,…).
For the calculations: a separate standard curve has the be performed for every independent experiment.When standard curve parameters are set, the cells of interest can be evaluated according to the mathematical approach described in detail in the guidelines section.