FACS analysis of receptor surface expression using fluorescent antibodies - (Mar/17/2006 )
I analized the surface expression of some receptors using FACS with fluorescent antibodies . I got peaks for control antibody- treated samples, recptor-specific antibody-treated samples. For each type of antibodies the samples were pre-treated with either the drug of interest or its control vehicle. But I do not know how to manipulate the results. I have now the following peaks:
1) peaks for control antibody- treated samples that were pretreated with vehicle
2) peaks for control antibody- treated samples that were pretreated with the drug of interest
3) peaks for recptor-specific antibody- treated samples that were pretreated with the vehicle
4) peaks for recptor-specific antibody- treated samples that were pretreated with the drug of interest
I need to know how can I say that the drug of interest increase (or does not affect) the sruface expression of the receptors? also how can we calculate the fold increase in receptor surface expression?
what is the recommended sources of information to understand data analysis for such technique?
comments are welcome and thanks in advance for cooperation.
People normally overlay flow cytometry plots and look for shifts in the staining or look at changes in the mean fluoresence intesity (m.f.i.) in the histogram statistic section (BD software). Your control antibody is just an isotype control antibody. Theoretically this shouldn't bind anything specifically so give you your background reading. Hopefully this staining wouldn't change with vehicle or your drug. So the m.f.i. won't change and the plots should superimpose each other when overlaid.
Your receptor ab should give a large shift in fluorescence when overlaid with the isotype control and a large increase in the m.f.i. Any effect of the drug (positive or negative) should be seen as shifts in the peaks when overlaid relative to the vehicle treated or changes in the m.f.i.
It might be worth doing a control without vehicle stained with the isotype ab and the receptor ab to make sure the vehicle has no effect on the cells either.
All the best,
I have done two experiments using FACS analysis of receptor surface expression stained with fluorescent antibodies. the results are described hereunder with the figures obtained are in the attached files.
In both fig. 1 and Fig.2, the white-color filled peak is for sample treated with the drug of interest and stained with control antibody (N.B., the peaks obtained from both drug and vehicle treatments and stained with control antibody are overlapping, so only one of them is shown here). While the empty peak outlined with red line represents sample treated with control vehicle and stained with receptor-specific antibody.
The green peak (In Fig.1):and the blue peak (In Fig.2) represent sample treated with the drug of interest and stained with receptor-specific antibody.
Although both the green peak and the blue peak are shifted to the right, their magnitudes are depressed.
My questions are:
1) can we consider such shift an indication of increased surface expression of the receptor of interest caused by drug treatment?
2) In both fig. 1 and Fig.2, the empty peak outlined with red line (that represents sample treated with control vehicle and stained with receptor-specific antibody) show a different pattern. In fig1 its superimposing the control antibody peak, while in fig2 its magnitude is depressed with slight shift to the right. Does this mean a particular thing?
3) how to quantitate such expression? Could you describe to me the steps to be followed to do this on BD bioscience software?
Thanks in advance
first i suggest you to put the FL2 scale in logarithmic mode for such a staining...
secondly, i see in the 2nd experiment that the control-treated cells expressed the receptor but at a lesser extend than in the drug-treated cells; on figure i see that non-treated cells don't express the receptor of interest at all...
but before verify that in the 2 experiments MFI(treated)/MFI(non-treated) are equal or not
you need a third experiment to give a correct conclusion!