1Understand multicolor flow cytometry control requirements00:05
2Prepare single stain controls for compensation00:44
3Apply fluorescence minus one controls01:38
4Use biological controls to detect non-specific binding03:10
5Select optimal controls for assay development04:28
Cell BiologyBD Biosciences
Choosing Proper Flow Cytometry Controls
Protocol
Difficulty
intermediate
Steps
1
Understand multicolor flow cytometry control requirements
Learn why multiple control types are necessary for accurate multicolor flow cytometry data interpretation, especially for dimly positive cells and avoiding misinterpretation of background signals.
▶ 00:05
2
Prepare single stain controls for compensation
Run individual antibody stains for each marker in the panel to calculate compensation and correct spillover signal from one fluorophore into another detector, avoiding false positive population identification.
▶ 00:44
3
Apply fluorescence minus one controls
Prepare FMO controls containing all antibodies except the marker of interest to account for background spread introduced by other fluorochromes and accurately set gates for positive populations.
▶ 01:38
4
Use biological controls to detect non-specific binding
Employ negative biological controls (unstimulated cells) and positive biological controls (stimulated cells with mitogens) to account for antibody-specific background noise that FMO controls cannot capture.
▶ 03:10
5
Select optimal controls for assay development
Run multiple control types during assay development to identify the main source of background in your specific experimental conditions and determine the best strategy to account for it.