Understanding FACs Readouts

[Keilza]

Hi there,

 

I just had a (hopefully) quick question about a couple of FACs readouts I've obtained after I treated some DCs with some antibody cocktails.

 

Basically, I understand the x-axis corresponds to size of cells and that the y-axis sows increases in granularity. However, I'm not certain what the %numbers in the corners of the different gates are supposed to mean or what the different dyes are supposed to represent?

 

Any kind of explanation to what I should actually be reading from FACs readouts would be much appreciated.

 

Thanks very much!

[MedGen]

The colours on the plots them selves aren't dyes. Everytime an event is recorded it is plotted based on its forward scatter and side scatter (the two left hand panels). Sometimes an event will occur in the same 2D position (that is at the same X,Y co-ordiantes) as another. Everytime an event occurs in the same position as another it changes the colour of the event on the plot. This goes from blue (few events) through light blue, green, yellow and then to red (many events). So the areas of red are essentially lots and lots of cells all of the same size and granularity and can be indicative of the purity of the population of that cell type. Not all cells will be exactly the same size or granularity but will tend to be around a given range.

 

The numbers in the gating look to indicate the % of events within that gate as a fraction of total events. For instance in the top middle panel the gating has determined that 1.8% of all events occur within that user-defined section (~340 events). This is confirmed by the panel to the right which is the gated section and tells you that there are 343 events within that gated section.

 

The far-most panels are a measure of the number of events (cells) within the gated sections corresponding to the intensity of that particular antibody (the x-axis is designated FL1-H) flurochrome.

Edited March 7, 2010 by MedGen
typo

[Keilza]

Thanks very much that's a great help to me.

 

For some reason it didn't occur to me what the dyes actually represented, so this has been very enlightening haha.