Flow Cytometry Troubleshooting Guide: How to Improve Flow Cytometry Analysis

Flow cytometry characterizes and/or sorts heterogeneous suspended cell populations based on physical and fluorescence characteristics. For optimum results and data analysis, a basic understanding of fluorochromes and their characteristics, spectral overlap and spillover, compensation and spread, as well as flow cytometer variables that can affect the sensitivity of the measurements is necessary. 

The following flow cytometry troubleshooting guide describes possible causes and solutions for most common problems encountered during flow cytometry experiments. Click on the common problems below for tips on how to improve your flow cytometry analysis, organized in easy-to-use tables.

• Acquisition Rate Decreases Dramatically
  
• A Loss or Lack of Signal
• High Background and/or Non-Specific Staining of Cells
• Variability In Results From Day to Day
• High Fluorescent Intensity
• Suboptimal Cell Scatter Properties
• Antibody Worked In Other Applications But Not In Flow Cytometry

We are committed to providing the best solutions and services to researchers around the globe. This flow cytometry troubleshooting guide does cover most commonly encountered problems. However, some problems may be related to one’s specific experiment. Please contact our Technical Service team for any specific issue you may encounter with your flow cytometric application of our antibodies. 

Read How to Select Flow Cytometry Dyes for more tips to enhance your flow cytometry experiments.

Acquisition Rate Decreases Dramatically

Cause	Suggested Solution
Blocked fluid pathway on the instrument	Reduce the number of cells being analyzed by diluting the sample to approximately 0.5 million cells per mL. Cell densities in excess can essentially block the normal flow, disrupting the assay. After many uses, it is possible that the fluid system on any standard flow cytometer will require cleaning. Run standard cleaning procedures to clean the fluid system during or after an assay. This will prevent any material from forming where the steady flow stream takes place.
Cells are clumping	Adherent or sticky cells can result in cellular clumping. Using a more aggressive enzyme for dissociation, such as trypsin, during cell harvesting should help keep cells in suspension. Alternatively, use a cell strainer to eliminate cell clumps.

A Loss or Lack of Signal 

Cause	Suggested Solution
Not enough cells in the sample	Cell loss is common during washing steps in the assay procedure. Make sure that cell density remains at approximately 0.5 million cells per mL during analysis.
Target is intracellular	Use an appropriate permeabilization protocol to ensure intracellular staining. Some cell fixative agents may compromise the detection of certain surface epitopes. Hence, it is best to test the fixative effect prior to the staining process.
Target protein is not expressed	Review the literature to confirm that the protein of interest is expressed in the cells being tested.   Make sure your sample contains cells that express a sufficient amount of the target protein. When targets are expressed at a low concentration or expressed in a low frequency, it is best to include a co-stain to differentiate low expressed target or low frequent cell population.
Conjugation kit failed	Try using a pre-conjugated primary antibody. Test the conjugation kit with a different antibody.
In a multicolor experiment, dye brightness and antigen expression are not optimally matched	Review the literature to check for antigen expression levels in the particular cell type. Use a bright dye with low expressing antigen. See Multicolor Flow Cytometry for more tips.
Cells may have to be stimulated to express a target protein	Some targets may be expressed only after stimulation with an appropriate agent. Try different stimulation agents, concentrations, and/or duration of treatment to optimize target protein expression.
Antigen-antibody binding may be suboptimal	Check for species specificity of the antibody. Optimize the antibody incubation time and temperature.
Antibody has degraded	Ensure that the antibodies are stored at the recommended temperature. Try a new batch of antibody.
Primary and secondary antibodies are not compatible	The use of a good secondary antibody can improve results significantly and eliminate or reduce false positive or negative staining. Use a secondary antibody that matches the class and/or subclass of the primary antibody. Use a secondary antibody raised against the species in which the primary antibody was raised. See How to Choose a Secondary Antibody for more tips.
Too many dead cells in the sample	Antibodies tend to stick to dead cells which can result in false positives. It is best to use a viability dye to assess cell health. Try a fresh preparation of cells.
Not enough antibody	A lack of signal may indicate that excess antibody will need to be used during the staining procedure. Further antibody titrations may be necessary for some cell types to capture the ideal staining concentration.

High Background and/or Non-Specific Staining of Cells

Cause	Suggested Solution
Too much antibody	Non-specific staining and background may indicate that less antibody will need to be used during the staining procedure. Further antibody titrations may be necessary for some cell types to capture the ideal staining concentration.
Cross-reactivity between secondary antibody(ies) and one primary antibody in your experiment	Select a secondary antibody that specifically binds to the primary antibody. Use a pre-conjugated primary antibody.
Non-specific cells are targeted	Include an appropriate isotype control and subtract any Fc binding signal. If needed, include a secondary antibody-conjugate control. Select a secondary antibody that does not display cross-reactivity. Pre-incubate cells with appropriate Fc blockers before staining to decrease non-specific binding of cells. Include 1-5% BSA in PBS when staining cells to decrease the non-specific binding of cells.
Too much secondary antibody	If a secondary antibody is used, try further reducing the amount of secondary antibody.

Variability In Results From Day to Day

Cause	Suggested Solution
Changes in cell viability	Monitor experimental cell cultures to ensure that the cell viability and cell numbers being analyzed are consistent.
Instrument calibration changes	Perform a quality check on the instrument (e.g., calibration) on a daily basis or prior to use.
Different batch of antibody used	Use one batch of antibody for each set of experiments. Use a pre-conjugated primary antibody.

High Fluorescent Intensity

Cause	Suggested Solution
High antibody concentration	Reduce the amount of antibody added to each sample.
Excess of antibody trapped	Perform adequate washing and include a detergent, such as Tween® 20 or Triton® X-100 reagents in wash buffer.
Too much secondary antibody	If a secondary antibody is used, try further reducing the amount of secondary antibody. Try pre-conjugated primary antibody. Use vendor-validated reagents to limit the amplifying effect of secondary antibodies.

Suboptimal Cell Scatter Properties

Cause	Suggested Solution
Incorrect instrument setting	Ensure that proper instrument settings are loaded.
Poorly fixed or permeabilized cells	Follow a standard fixing and permeabilization protocol. Avoid damaging cells by rapid immersion of cells in hypotonic solution or methanol.

Antibody Worked In Other Applications But Not In Flow Cytometry

Cause	Suggested Solution
Antibody was not suitable and not recommended for flow cytometry	Use an antibody that has been tested and recommended for flow cytometry applications. Contact Technical Service through the web here, or call them here.