FMO Protocol

What is FMO:

FMO stands for Fluorescence Minus One, this is a gating control. Contrary to popular belief or historical wisdom Isotype is NOT a gating control. Correct use of Isotype control is to find out how much blocking reagent is needed. If we design the panel properly usually, we do not need FMOs or may need one.

We use FMOs only if we are unsure where the positivity starts in the plot. As shown in this image. In the fully stained sample, it was not clear from where CD45RA-PE positivity begins. With the FMO in the middle it is easy to identify where positivity starts, shown by the blue dotted line.

Like FMO there is another concept of Fluorescence Minus TWO where we can choose not to add two abs instead of one, provided these two colors have no bleed through or cross talk.

How to make FMO:

One FMO for a panel: Let’s assume the panel has 10 color/Abs. We need to create a FMO for color number 5. We are simply going to create a master mix of 9 color/abs EXCEPT color 5. Immediately after adding this master mix to the cell (with proper blocking) we will take a small portion aside; this is going to serve as FMO for color 5. Then we will add required volume of Ab 5 to the main bulk of the cell, and this will become the full stain. Both samples will be the allowed to stain for certain time and washed in similar manner.

Two or multiple FMOs for a panel: Let’s assume the panel has 20 color/Abs. We need to create two FMOs for color number 7 and 12. We are going to create a master mix of 18 color/abs EXCEPT color 7 and 12. Will add this master mix to the cell and take two small portions out, these will be used to prepare the FMOs following the below table.