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Flow Cytometry Sample Preparation

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Flow cytometry sample preparation

As in any antibody-based technique, biological samples require preparation prior to staining and analysis. Cells require disassociation and are often fixed and permeabilized when analyzing cytoplasmic and nuclear proteins. Samples need to be washed after staining to remove any excess of unbound fluorescent reagents. Please refer to our experimental workflow (Figure 4) and sample preparation table (Table 3).

Figure 4. Experimental workflow for analyzing protein targets in flow cytometry

 

Table 3. Sample preparation steps in flow cytometry experiments.

Sample preparation step Description and tips
Cell dissociation
  • Adherent cells need to be detached prior to flow cytometry analysis using detaching agents, e.g., trypsin. Milder enzymes are frequently used for cell detachment in order to preserve plasma membrane receptors if they are a subject of analysis.
  • Use appropriate buffers to prevent cell clumping – avoid calcium and magnesium salts. If they are present, a chelating agent, e.g., EDTA, can be added to samples.
  • Tissue samples require more stringent disassociation methods – either mechanical or enzymatic (e.g., trypsin, collagenase).
Fixation
  • Advantageous for sample preservation but can increase autofluorescence levels.
  • Two types of fixatives available: alcohols (methanol or ethanol) and aldehydes (formaldehyde or glutaraldehyde).
Permeabilization
  • Necessary for staining of intracellular proteins.
  • Mild detergents, such as saponin or digitonin, are used for analysis of cytoplasmic proteins.
  • Harsher detergents, e.g., Triton X-100 or NP-40, need to be used for analysis of nuclear proteins to ensure permeability of both the plasma membrane and the nuclear envelope.
Incubation with fluorophores, dyes, or antibodies
  • Optimization of used concentrations needed to achieve best signal to noise ratio and to reduce non-specific binding.
  • Choice of dyes influences other steps (e.g., compatibility with used fixatives).
  • Careful design needed for multicolor analyses (see section 4).
Washing
  • It is necessary to remove remnants of fixatives, permeabilization agents, and unbound fluorophores.

The proper execution of sample preparation steps helps to reduce noise and ensure good-quality data are obtained. The presence of many dead cells, cell clumps, and high cell autofluorescence can usually be avoided. For example, the presence of dead cells can be spotted by analyzing FSC vs SSC plots – dead cells have low FSC and high SSC compared to live cells. It is good practice to include viability dyes in experiments as this provides a definite method of excluding dead cells from analysis. Dead cells are particularly sticky to antibodies and can therefore significantly influence and falsify results if they are not excluded.

It is also important to optimize the dilutions of antibodies and other probes used for staining. This not only allows the saving of reagents but also reduces the background from non-specific binding. The correct dilution depends on the antibody affinity to the target and target abundance. Very high dilutions are recommended for abundant targets. As seen in Figure 5, it is possible to easily discriminate between CD4-positive and CD4-negative lymphocytes stained with even very high dilutions of the anti-CD4 antibody.

Figure 5. Dilution of antibodies for sample staining in flow cytometry.

1X10^6 human peripheral blood lymphocytes were surface-stained with APC-Mouse IgG2b isotype control (1) or APC-Anti-Human CD4 (APC-65134, clone OKT4) using 2-fold serial dilutions (2-10). Samples were not fixed.