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Optimizing your flow cytometry staining protocol

Figure 18. Some of the required controls for flow cytometry.

When setting up your flow cytometry test, it is important to consider cytometry controls, which are essential for ensuring accurate and reproducible results in flow cytometry experiments.

Make sure you include the appropriate controls for:

• Cell viability: Dead cells can produce artifacts due to non-specific binding and autofluorescence, potentially leading to inaccurate results

• Autofluorescence: Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence that may mask antigen-specific signals

• Spectral overlap: Fluorescence emitted from one fluorophore may also be detected on a different channel, significantly affecting measurements on a given channel. Compensation controls, including the use of compensation beads, are used to correct for spectral spillover into other channels and ensure accurate gating.

• Undesirable antibody binding: This occurs when the antibody binds to either an off-target epitope, Fc receptor (FcR), or cellular components through its conjugated fluorophore. FcR binding can be reduced with the addition of specific blocking reagents before staining

Including essential controls and proper controls is critical for obtaining reliable, publication-quality data in flow cytometry experiments. These controls help ensure accurate data interpretation and reproducibility in all flow cytometry experiments.

Cell viability

It is essential to eliminate dead cells from your flow cytometry data analysis because they can give rise to false positives due to autofluorescence and increased non-specific binding.

Several markers are available that can distinguish between dead and live cells. As some of these dyes bind DNA, they may also be used for DNA content or cell cycle analysis.

Cell impermeable dyes such as 7-Aminoactinomycin D (7-AAD), propidium iodide, Nuclear Green DCS1, or DRAQ7™ are used on unfixed cells. These dyes discriminate between live and dead cells by staining only dead cells and being actively excluded from living ones.

An alternative method for determining cell viability is to use the cell-permeable fluorescent dye calcein AM. This dye is hydrolyzed to green fluorescent calcein by intracellular esterases in living cells. Cells stained with this dye can also be fixed with paraformaldehyde and then analyzed.

Autofluorescence

Cell type and physiological conditions influence autofluorescence. Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence upon 488 nm wavelength laser excitation.

To check if autofluorescence presents a problem in your experiment, analyze an aliquot of unstained cells on the flow cytometer using the same cell treatment and machine settings as the experimental sample. If there is significant autofluorescence, using a different laser wavelength can resolve the problem.

Refer to our fluorophore chart or multicolor selector to explore alternative lasers using your dye of choice.

Spectral overlap

When carrying out a multicolor flow cytometry experiment, the emission spectra of the various fluorophores can overlap, resulting in detection in a different channel (also called spillover) and affecting other channels. However, this can be controlled with compensation, where spectral overlap is estimated and subtracted from the total detected signal to yield an estimate of the actual amount of each dye. Compensation controls can be set up using single stained samples or compensation beads, which are synthetic beads that bind to conjugated antibodies and serve as a reliable, non-specific tool without needing actual cells.

Additionally, including fluorescence minus one (FMO) controls, such as a PE FMO control for the PE channel, helps define the positive/negative populations and improves the reliability of gating strategies.

FMO controls

FMO controls, samples stained with all antibodies in a panel except for one, are essential for providing a measure of spillover in a given channel and accurately discriminating positive and negative cell populations in a multicolor experiment¹. This control provides a true negative control as it considers how the other fluorophores in your panel affect the signal observed in the channel used for the examined fluorophore. FMO controls help to accurately identify the positive population, positive populations, and negative population within a cell population by accounting for fluorescence spread and background. For example, in a multicolor panel of FITC, PE-Cy5, PE-Cy7, and PE, the PE FMO control would contain the FITC, Cy-PE, and Cy7-PE reagents, but not the PE (Figure 19).

Figure 19. Example of an FMO control. If an unstained control was used to set the gate for PE positive/negative cells (lower line, unstained gate), all cells above this line in the right-hand dot plot would show as positive for PE. However, when using an FMO control to set the gate (upper line, FMO gate), it becomes clear that there is a spread of signal, and some cells are not positive for PE.

Undesirable antibody binding

This broad term includes every instance of antibody binding, including nonspecific binding, that prevents correct interpretation of the data. Optimizing your staining protocol and running appropriate negative controls can help to detect and alleviate these effects. Using a secondary antibody control is essential to assess nonspecific binding in indirect immunostaining and flow cytometry. Additionally, it is important to match isotype controls to the experimental antibody in terms of species, isotype, and conjugation to ensure specificity and accurately interpret results.

Optimizing your multicolor flow cytometry staining protocol

Antibody validation

Antibodies are key components of flow cytometry techniques, yet many have not been validated for specificity, lack of cross-reactivity, or use in flow cytometry applications.² To ensure reproducible, robust data, it is important to either validate your antibodies or purchase validated antibodies from a trusted supplier.

Antibody concentration

Non-optimal antibody concentrations can increase non-specific binding or reduce the sensitivity of the measurement. Therefore, titrate all antibodies to determine the best signal-to-noise ratio (Figure 20).

Figure 20. Titrate antibodies to improve sensitivity in flow cytometry.

Fc blocking

Phagocytic cells, such as monocytes, have Fc receptors (FcRs) on their surface that can bind nonspecifically to the Fc region of antibodies—adding FcR blocking reagents before staining can block this binding (Figure 21). You should also include this blocking step in homogenized tissue samples, which may contain macrophages, as well as cell culture lines such as Daudi and THP-1.

Figure 21. Fc blocking to reduce non-specific antibody binding in flow cytometry.

Negative controls

The negative control should be a population of cells that do not express the antigen of interest, ideally a knock-out cell line or other biological controls. Biological controls serve as negative or positive references to confirm staining specificity and evaluate assay performance. Negative controls can also include experimental cells with a known marker expression status, such as cells confirmed to lack the marker of interest. This sample should be exposed to the same experimental conditions as the population in the study. Use this control to set gating regions and distinguish cells lacking the marker of interest from those that are positive.

Isotype controls

An isotype control is an antibody raised against an antigen not present on or in the analyzed cell type. Isotype controls determine the level of background fluorescence caused by non-specific antibody binding. They should not be used to distinguish positive from negative cells or set positive gating regions.

An ideal isotype control should:

• Match the primary antibody in host species, class and subclass of heavy and light chains, fluorophore type, and number of fluorophore molecules per immunoglobulin
• Be derived by the same manufacturing process and presented in the same formulation

Refer to our guide to selecting isotype controls for more information here.

Isoclonic controls

The isoclonic control shows whether a fluorophore or other antibody conjugate is binding non-specifically to cellular components.

Cells are stained with the conjugated antibody in the presence of an excess of identical (isoclonic) unlabeled antibody. Specific antibody binding sites in the sample are taken up by the unconjugated antibody, while the conjugated antibody can only bind through the conjugate.

A lack of fluorescent signal suggests that the conjugate is not binding non-specifically to any components within the sample. As with any isotype control, this type of control is solely qualitative.

References

1. Nguyen, R.,, Perfetto, S.,, Mahnke, Y.D.,, et al. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design Cytometry A 83 (3),306-315 (2013)
2. Kalina, T.,, Lundsten, K.,, Engel, P. Relevance of Antibody Validation for Flow Cytometry Cytometry A 97 ,126-136 (2020)