Hi Eman,

The answer depends on if you want a quantitative or qualitative level of expression. For quantitative, you will need qPCR, Western blot, or a fluorimeter/plate reader against a standard with known concentration.

For qualitative, you can compare to another cell line expressing eGFP at a known amount. In that case you can use any method you like as long as the parameters are constant between the known and unknown samples.

Hope this helps!

I suppose you want absolute quantitive data, because relative quantitative data can obviously be acquired by any device that provides (reasonably linear) output on fluorescence intensity, such as a fluorometer, fluorescence plate reader, or a fluorescence microscope equipped with a digital camera.

It depends a bit if you want expression quantified at a single cell level or just at a cell population level, in other words, if it sufficient for you to know the mean eGFP expression or if you require a distribution of expression levels.

For the population only level expression level proceed as follows:

1) Purchase known amount of eGFP.

2) Prepare samples with a known number of non-transfected cells and eGFP-expressing cells. [E.g. five samples of the non-transfected cells and your transfected sample.]

3) Lyse the cell batches from the phase 2 with a nondenaturing lysis buffer.

4) Add your purchased eGFP to the samples with non-transfected cells in varying amounts to produce a standard curve.

5) Measure fluorescence intensity from the cell lysates (with added eGFP and from your unknown).

6) Use the standard curve to establish the amount of eGFP in your eGFP-expressing cell sample.

For the single cell level expression (i.e. the distribution of expression), the method of choice is obviously flow cytometry with your cells and with microspheres containing known amounts of eGFP (e.g. http://www.ebiotrade.com/emgzf/clo2002apr/FACS-EGFP.pdf). Then you get wonderful statistics easily. If you do not have access to a flow cytometer, then you have two options (A & B) available with a fluorescence microscope. AT ALL POINTS, BE SURE TO AVOID SIGNAL SATURATION.

1A) Nevertheless purchase the eGFP-microbeads, 1B) purchase just eGFP.

2A) Image (preferably) hundreds or microbeads of each intensity set with a fluorescence microscope. Segment microbeads from the background. Use the particle area-integrated(or in confocal volume-integrated) fluorescence intensities to recover the distribution of fluorescence intensities of the microbeads. Create a standard curve. 2B) Do the population only level expression measurement described earlier to recover the mean expression level in your cell population.

3AB) Image hundreds of cells from your eGFP-expressing cell population. Define the cells in images, calculate area-integrated (or for confocal volume-integrated) intensity for each cell. Then 3A) match the intensities to the standard curve from the mean intensities of microbeads, or 3B) match the mean intensity to the mean intensity of the population experiment, and assume linearity of the intensity readings.

Thanks Andrew for your answer. In fact, I am trying to compare different promoters for expression eGFP, which is better. Unfortunately, I don't have an expressing model with a known level. But I will follow your suggestions for the future.

Thanks again!

Thanks Juha-Matti for your detailed answer. Yes, I am using fluorescent microscope at the moment and I need to determine the strength of different promoters. I will try do what you suggested for future work.

Thanks again!

Well, if you just want to check the efficiency of different promoters for GFP expression, then you do not need absolute quantitation, you just need relative amounts. It is sufficient that you can say that promoter A leads to cells expressing twice as much GFP as promoter B, and you do not need to know at all how much GFP either A or B is expressing. If it is not a secreted construct but one that remains inside the cells, then you just transfect the cells in triplicate, treat them otherwise just the same, along with non-transfected controls, then take fluorescence images and brightfield images from all the populations (including non-transfected controls, and with fluorescence and BF from the same spots), segment the cells from the background (from the brightfield image so that you will not miss the cells that are not expressing GFP) and integrate the fluorescence intensity and count the number of cells, then you can get the mean fluorescence intensity per cell in each transfected population (and from each cell in each population as well). Calculate the mean and standard deviation from your triplicates and see if they are different. As a bonus you get the distribution of expression too, so it's like doing single cell qPCR for GFP, so much better than doing ordinary qPCR. In fact it is even far better than doing single cell qPCR for the GFP construct, because I suppose, you, too are interested in the expression of the protein construct, not the expression of mRNA that gets destroyed before translation.

Just take care that you are not overexposing the images, you do not want to have cells or regions of cells that have the maximum fluorescence intensity of your camera.