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Tips for selecting controls and preparing samples for flow cytometry.
Flow cytometry is a valuable technology in modern biomedical research. It is widely used for the characterization of cell populations and expression analysis of protein markers. However, poor experimental design may render your results useless. This article is dedicated to provide you with valuable tips to set up your experiment and prepare your samples.
|Control||What to include||Purpose||Comments|
|Unstained control||Cells that are fully processed without addition of any antibodies.||To measure autofluorescence. May be used as an additional negative control.|
Comparison to beads can help to determine the relative amount of autofluorescence. Consider using a different excitation source if autofluorescence levels are high.
|Internal negative control||Population of cells that do not express the antigen of interest and are fully processed.||To avoid false positives resulting from nonspecific antibody binding.|
Cells to use as a negative control might not be always available. Ideally, the fluorescence intensity of the internal control should be the same than the unstained control.
|Isotype control||Cells incubated with isotype control antibodies (antibodies usually raised against an antigen that should not be present in your cells).||To determine nonspecific binding of the primary antibody.|
The isotype control should match, at least, the heavy chain (IgA, IgG, IgD, IgE, or IgM) of the primary or secondary antibody, and be conjugated to the same fluorophore.
|Secondary antibody control||Cells incubated only with the secondary antibody.||To determine nonspecific binding of the secondary antibody.||Only necessary if using a secondary antibody.|
|Positive control||Cells known to express your target of interest.||To avoid false negatives resulting from a faulty antibody.|
Positive control cells might not be always available.