10 Tips for successful flow cytometry

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With this in mind, here is a list of tips compiled by Dr. Stephanie McArdle and Prof. Graham Pockley from the John van Geest Cancer Research Center (UK) for achieving high quality data by flow cytometry, a method allowing the analysis of the physical and chemical properties of cells and other particles such as nuclei, chromosomes or small beads in a flow system as it passes through at least one laser. Most importantly, it holds a huge potential as it can be used for a wide range of applications such as multi-color analysis, cell viability, cell cycle analysis, apoptosis/necrosis, functional analysis (calcium flux, cell proliferation, autophagy), rare event analysis (dendritic cells, stem cells/progenitor cells, memory cells), organelle function etc.

1. Sample preparation

All experiments start here. It is therefore essential to optimize sample preparation, especially when analyzing adherent or tissue-derived cells following harvesting. The procedures used should not negatively influence cell viability, and the use of enzymatic isolation procedures should not have any effect on the expression of the markers that are being studied.

2. Eliminate non-specific binding

It is essential to prevent non-specific binding of antibodies to the cells in the sample. This can be achieved by incubating samples with serum from the same species as the antibodies that are being used (10% v/v for 10 minutes prior to staining) or by using dedicated blocking solutions (e.g. FCR block). This is especially important if the analysis is focused on monocytes or dendritic cells, as these cells can express a range of Fc receptors that can bind antibody via their Fc region, rather than via their antigen-specific binding site.

3. Titrate your antibodies

It is essential to titrate all antibodies in order to ensure that an appropriate amount is being used for each experiment. Too much antibody could increase non-specific binding, and too little antibody could mean that you are unable to detect the presence of the target antigen. Although antibodies can be titrated singly, it is possible that the optimum concentration is different when the antibody is used as part of a panel. Fluorescence Minus One (FMO) staining should therefore be performed in order to assess the amount of ‘spill-over’ into one particular detector and confirm the population boundaries. This is a “must” if dealing with a dim or non-discreet population and if evaluating spill-over containing discreet lineage markers. In other words, if the population analysed are clearly distinct from one another then there is no need for the FMO. The FMO is an essential component for ensuring the correct instrument set-up and compensation (see below).

4. Detection and elimination of dead cells

Dead cells can be a source of non-specific staining especially if the cells are being used after having been thawed from frozen. They can be detected by using a DNA dye which would only enter cells if their membrane integrity has been compromised. A reliable fixative will be needed if intra-cellular staining is to be performed thereafter.

5. Detection and gating out doublets

Doublets (when two cells are stuck together and are analyzed as one) will falsely increase the fluorescence intensity of the ‘cells’ passing through the laser interrogation point. They can be easily gated out by creating a window using the Forward Scatter INT versus Forward Scatter Time-of-Flight (ToF) or Side Scatter INT versus Side Scatter Time-of-Flight (ToF). Following on from this, it is important to filter the cells before analyzing them in order to exclude cell clumps.

6. Dye stability

Please be aware that tandem dyes can degrade with time, light and heat exposure. This can give you a false negative in the correct channel and a false positive in the channel which is relevant to the original fluorochrome. For example, PE and APC tandems can spill-over into the PE and APC channels. It is therefore essential to store your reagents correctly and keep your samples protected from light.

7. Choice of fixation and permeabilization reagents

Certain intra-cellular antigens can be difficult to stain. The choice of fixative and permeabilization solutions is important and will depend on the antigen which is being examined. It is therefore essential to optimize the staining protocol using available reagents or the manufacturer’s recommended reagents. In summary, not all fixation and permeabilization solutions work the same in all experimental settings.

8. Choice of fluorochrome

You should know your instrument configurations (lasers, filters) and the excitation and emission spectra characteristics of the fluorochromes that you are considering for your experiments. It is impossible to design a successful experiment without having this information. You should also be aware of the ‘brightness’ of the different options as typically, one should use a bright fluorochrome to detect antigens that are expressed at low density, and dim fluorochromes to detect antigens that are expressed at high density. However, the choice of fluorochrome is also important for compensation, and the possibility that ‘bright’ fluorochromes might cause problems with spectral overlap should be considered.

9. Compensation

You should be aware that the more fluorochromes are being incorporated into an experiment, the more complicated this experiment will be when it comes to minimizing spectral overlap and the ‘leakage’ of fluorescence between different channels. One needs to consider issues such as the loss of sensitivity, reagent interactions, and unwanted energy transfer between adjacent fluorochromes. If possible, one should spread the fluorochromes over multiple lasers in order to minimize both spectral overlap and compensation. Compensation controls can be set up using beads, rather than precious samples.

10. Sample storage

Ideally, samples should be analyzed immediately. However, if this is not possible, samples can be resuspended in 100µl fixative (e.g. 1-2% formaldehyde), or other commercially-available solutions, and stored at 4°C, protected from the light for up to 24 hours (or even longer if optimized).

Although these are some tips, there are clearly many other issues that need to be considered and addressed in order to ensure a successful experiment, such as correct data acquisition and analysis approaches, standard buffer and sample preparation procedures etc. However, the key thing is to understand the principles and practice of flow cytometry, and be aware of the pitfalls and limitations of this approach for detailed cell analysis.

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