Flow cytometry – experimental design and sample preparation

​​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.

Experimental design - a thorough approach will pay off

  • Research what is available in the literature, specifically in relation to your sample and experiment. Groundwork will avoid mistakes further down the road.
  • Make sure your instrument is suitable for the detection of the fluorophores you decide to use. You want fluorophores with maximum brightness and minimum spillover. Try our new Alexa Fluor® conjugated secondary antibodies validated for flow cytometry.
  • Make sure you have all the reagents you need in excess. Prepare a step-by-step protocol and calculate the amounts of reagents required for your number of samples.
  • You may want to use a marker of dead cells as their presence can significantly affect your analysis. Various assay-compatible dead cell markers are now available.
  • Titrate your antibodies to find out the optimal dilution and minimize background. The working dilution provided in the datasheet can be used as a starting point.
  • Do your research to determine what to expect. For instance, cell lines are relatively homogenous while primary cells usually consists of a heterogeneous population of different cell types.


ControlWhat to includePurposeComments
Unstained controlCells 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 controlPopulation 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 controlCells 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 controlCells incubated only with the secondary antibody.To determine nonspecific binding of the secondary antibody.Only necessary if using a secondary antibody.
Positive controlCells known to express your target of interest.To avoid false negatives resulting from a faulty antibody.

Positive control cells might not be always available.

Sample preparation

  • Use BSA or FBS as a blocking agent to minimize non-specific binding.
  • Use Ca/Mg++ free buffers to prevent cation-dependent cell adhesion. You can add up to 5 mM EDTA to further prevent cell adhesion. Under these conditions, BSA (0.1 – 1%) or dialyzed FBS (1 – 5%) should be used as non-dialyzed FBS would replace the Ca/Mg++.
  • If studying intracellular markers, low concentrations of non-ionic detergents (up to 0.1%) are suitable to permeabilize cell membranes.
  • Inclusion of 10% homologous serum or 5 mg/ml unlabelled IgG during sample staining will minimize antibody binding to Fc-receptors.
  • For live cells, internalisation of cell surface proteins can be prevented by performing all steps on ice. Chill reagents at 4°C before use. Gentle detachments methods may be required for adherent cell lines as trypsin can induce internalisation of cell surface proteins.
  • Adding DNAse I (25 – 50 µg/ml) and 5 mM MgCl2 will prevent clumping due to cell death.
  • It is very important to obtain a single cell suspension to avoid clogging up the system. Filter your samples before the run with cell-strainer caps if possible. Use a nylon mesh only as a last resort as you may lose many cells with this system.
  • Keep cells at a reasonable concentration (1 x 106 – 10 x 106 cells/ml). Higher cell concentrations might clog up the system and affect resolution.
  • Prevent damage of the cells by avoiding bubbles, vigorous vortexing, aspirating the entire solution during buffer exchange, and excessive centrifugation.