I have transfected cells and the protein is expressed with GFP marker. when I want to check the expression by flow I see two peaks of the cell population: bright and less bright. I have a uniform cell population of 3T3 cell lines. What is the reason for the two peaks to occur?

omid said:

I have transfected cells and the protein is expressed with GFP marker. when I want to check the expression by flow I see two peaks of the cell population: bright and less bright. I have a uniform cell population of 3T3 cell lines. What is the reason for the two peaks to occur?

I am not totally clear on the question.
Do the transfected cells have 2 peaks total?
expression and no expression (transfection efficiency)
Or do the transfected cells have 3 peaks?
no expression, low expression and high expression?
I have observed a great variety of expression levels in GFP transfected cells resulting in long plateaus on histograms, but not distinct populations (peaks) of no, low and high expressing cells.

If youu just see two peaks in your plots (one that you call low, and another high), it is likely that the low population is untransfected - what you are seeing is the "cells alone" autofluorescence. This can be easily checked by running a control untransfected cell population from the same thaw/exp. set. Also, remember to plot these kind of data on log Fl1 axis, not linear: this makes differences more clear-cut.

samm said:

If youu just see two peaks in your plots (one that you call low, and another high), it is likely that the low population is untransfected - what you are seeing is the "cells alone" autofluorescence. This can be easily checked by running a control untransfected cell population from the same thaw/exp. set. Also, remember to plot these kind of data on log Fl1 axis, not linear: this makes differences more clear-cut.

We had the same problem that Omid is asking about. We checked everything and we found out that our cell line was contaminated with another line that had the same expression with different level, giving us two different shifts. We thawed a new tube from a much earlier frozen stock and the double shift disappeared.

Dear Omid,

as someone who does lots of cell cloning, let me pass on some words of wisdom.

Your transfected pool of cells contains many populations of cells. Some do not express your protein-of-interest (and will be seen as either a separate peak to the left or as the left-hand tail of the flow histogram). Some will express low, med and some high levels of your protein.

That is why generally flow histograms are gaussian (I hope that is spelt right).

If you have two very distinct peaks, then you have two very distinct populations of cells. Note the percent of positive cells. If the percentage is low and this is a problem, sort them. It is very easy.

Also note that over time in culture, the percent of positive cells can decrease and this will often be seen as the formation of a negative peak/population. As Roudi says, go to an earlier stock. This is why preparing a good source of liquid nitrogen backups is so important shortly after stabilising your transfectants.

Good luck with your cells.