Flow Cytometry
FMO Control in Flow Cytometry Experiment
Source: Elabscience®Published: Oct 16,2024
In experiments, we often use blank control, single positive control, isotype control, biological control, etc., but the use of FMO control is relatively rare. Despite this, FMO control still has important reference value when setting gate.
On the one hand, FMO control helps to accurately set the boundaries of positive and negative populations. When we do multi-color experiments, some channel signals may be difficult to distinguish, mainly because of the interference of another fluorescence on the channel. FMO control (Fluorescence Minus One) reduces the fluorescence antibody of one channel, helping to measure the fluorescence leak of other channels.
As shown in the figure below, the single-positive tube of PerCP/Cyanine5.5 has a clear grouping, while the full-stained tube cannot be grouped. Through FMO control (excluding PE from multi-color results), the channel signal of PerCP/Cyanine5.5 has a clearer grouping, which proves that PE has a greater interference on the PerCP/Cyanine5.5 channel (different instruments have slightly different filters, which may lead to differences in interference among different channels, please analyze based on specific instrument channels).
The other hand, FMO can serve as a negative control for certain markers, excluding the background effect of full staining, making gate setting more accurate.
As shown in the figure, if a blank tube is used to determine the negative cell population, the gray line is usually used as the negative cell gate for PE, which may result in a bias in the experimental group results.
If an FMO control is added, the PE positive and negative grouping moves up to the red line, making the gate setting data more accurate.
Other considerations:
● To ensure the accuracy of gate setting, the FMO control group needs to be done using the same cells, to ensure that the experiment is not interfered with for other reasons. Therefore, microbead compensation cannot be used as a substitute.
● If the experimental sample needs to be fixed, the FMO should also undergo the same procedure.
