Flow cytometry training

Part 2: sample preparation and staining

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Welcome to our training series on flow cytometry. We begin with essentials like getting to know your cytometer, before taking you through some of the finer points and ending with how to get the most from this powerful technique.

In Part 2 of flow cytometry training, we cover such essential steps as preparing samples, setting up controls, choosing between direct and indirect staining, and performing intracellular staining.

Part 2 overview

2.1 Sample preparation
2.2 Controls
2.3 Direct vs indirect staining
2.4 Intracellular staining

2.1 Sample preparation

Your sample preparation can have a considerable influence on the quality of your data. Watch our short video to find out why preparing a single cell suspension is so important for flow cytometry and how to do it.

Below are some top tips for sample preparation for flow cytometry:

• Minimize non-specific binding - use BSA or FBS as blocking agents.

• Minimize antibody binding to Fc-receptors - include 10% homologous serum or 5 mg/mL unlabeled IgG during sample staining.

• If studying intracellular markers, permeabilize cell membranes - use low concentrations of non-ionic detergents (up to 0.1%).

• If using live cells, prevent internalization of cell surface proteins - perform all steps on ice and chill reagents at 4°C before use.

• Prevent cell damage - avoid bubbles, vigorous vortexing, aspirating the entire solution during buffer exchange, and excessive centrifugation.

• Prevent clumping due to cell death - adding DNAse I (25–50 µg/mL) and 5 mM MgCl₂.

• Prevent cation-dependent cell adhesion - use Ca²⁺/Mg²⁺-free buffers. You can add up to 5 mM EDTA to further prevent cell adhesion. Under these conditions, you should use BSA (0.1–1%) or dialyzed FBS (1–5%) as non-dialyzed FBS would replace the Ca²⁺/Mg²⁺.

• Use single-cell suspensions - filter samples with cell-strainer caps if possible. If not, a nylon mesh can be used but you may lose cells this way.

• Prevent clogging up the system - keep cells at a reasonable concentration (1 x 10⁶–10 x 10⁶ cells/mL).

2.2 Controls

Proper controls will help you to distinguish between true and false results and identify potential problems in the methodology. So, you must take the time to set them up properly.

When setting up your experiment, make sure you include the appropriate controls for the following:

Cell viability

Dead cells can produce artifacts due to non-specific binding and increased autofluorescence levels, potentially leading to erroneous conclusions.

• Use viability markers (eg 7-Aminoactinomycin D (7-AAD), propidium iodide, Nuclear Green DCS1, or DRAQ7™) to distinguish between live and dead populations.

Autofluorescence

Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence that may mask antigen-specific signal.

• Use an aliquot of unstained cells to control for autofluorescence. You can also use unstained cells for defining negative populations, cell size, and granularity.

Spectral overlap

Fluorescence emitted from one fluorophore can be detected on a different channel. This phenomenon can seriously affect measurements on a given channel.

• Fluorescence-minus-one (FMO) control measures spillover in a given channel. Here, a sample is stained with all the fluorescent conjugates except the one that is being tested, highlighting the contribution of the other fluorescent conjugates in the signal of the unlabeled channel.

• The FMO control is fundamental in a multicolor experiment because it allows for correct gating and selects only the stained cells in the experimental sample.

• See our detailed compensation guide for additional information.

Undesirable antibody binding

This broad term includes every instance of antibody binding that prevents the correct interpretation of the data. Undesirable antibody binding occurs when the antibody binds to either an off-target epitope or an Fc-receptor (but not as a receptor-ligand interaction) or binds through to an epitope or antigen through its conjugated fluorophore.

• Use negative and isotype controls to account for this.

• The negative control should be a population of cells that do not express the antigen of interest; use knockout cells if possible. This sample should be exposed to the same experimental conditions as the population in the study. Use a negative control for setting gating regions and discerning positive from negative cells.

• Isotype controls are used to determine the background caused by non-specific antibody binding. An isotype control uses an antibody of the same isotype as the primary antibody but that is specific for an antigen absent from the cells under study. Isotype controls should be used to determine the background due to non-specific antibody binding. They should not be used to distinguish positive from negative cells or set positive gating regions.

See our full list of recommended controls.

2.3 Direct vs indirect staining

Direct and indirect essentially refer to the location of the detection molecule, ie the fluorophore. For direct flow cytometry methods, the fluorophore is conjugated directly to the primary antibody. In indirect flow cytometry methods, the fluorophore is conjugated to a secondary antibody which binds the primary antibody – this can serve to amplify the signal at the expense of additional protocol steps. In this video, we’ll take you through the main steps of both direct and indirect flow cytometry protocols.

Download our flow cytometry protocols.

2.4 Intracellular staining

While commonly used to detect cell surface markers, like CD proteins, flow cytometry can be used to examine intracellular proteins, such as transcription factors, as well. To do this, you need to make use of both fixation and permeabilization steps before staining your cells.

For more details, refer to our intracellular staining protocol.

Summary

With Part 2 complete, you should now have a better understanding of:

• Key points in sample preparation
• The various types of controls and how to implement them
• Direct vs indirect staining
• How to modify flow cytometry protocol to look at intracellular proteins

In Part 3, we’ll guide you through an experimental setup for flow cytometry, compensation, and data analysis.

Start Part 3 now!