Flow Cytometry Troubleshooting Guide



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TROUBLESHOOTING

 


Flow Cytometry Issue observed Potential source and recommended solution

What to consider if no signal or weak fluorescence intensity is detected

If your signal is weak, your detection antibody may be too dilute. Although a primary antibody is validated for use in flow cytometry, a titration of the antibody concentration may be required for your specific cell or tissue type, or experimental conditions. Learn more about antibody basics.
Ensure that rare proteins are paired with bright fluorochromes. Use our Multicolor Panel Builder to adjust your panel as needed.
If fixation and permeabilization protocols are appropriate for the target in question and the optimal antibody titer has been determined for the specific experimental conditions, verify whether any pre-treatment of cells (e.g. stimulation of immune cells) is necessary to induce or augment the expression of the molecular target.
If no signal is detected, your target may be inaccessible. Check the predicted location of the protein and that fixation and permeabilization methods were properly used for the target of interest. To prevent the internalization of surface antigens, cells can be kept on ice during processing. In some cases, staining can be optimized by adjusting the incubation temperature or staining duration.
If secreted proteins are targeted, be sure that inhibitors such as Brefeldin A    or monensin were used. These compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport machinery, ultimately trapping the proteins in their respective cellular compartments. These inhibitors are required when assessing molecules such as cytokines.
With adherent cells where trypsin was used to dissociate cells from the surface, a source of weak signal can be related to trypsinization effects on the expression of extracellular molecules. Sodium azide prevents the modulation and internalization of surface antigens. If using cryopreserved cells, check whether the target antigen may be affected by freezing and/or thawing procedures.
Was the correct laser and filter combination used? Check the excitation and emission spectra for the fluorochrome used. Be sure that all lasers are well aligned, as misalignment can result in weak signals. The use of calibration beads can help decipher instrument performance for each channel.
Excessive light exposure during the staining procedure can result in photobleaching of fluorochromes and dissociation of tandem dyes. Make sure that samples are protected from light as much as possible. Tandem dyes can also be affected by fixation agents, especially if exposed to them for extended amounts of time. Remember to treat your single-stained compensation tubes the same as your experimental samples in order to control for any potential alteration to fluorescent properties from experimental procedures.
For very low cell density or poorly expressed intracellular targets, techniques like Single-Cell Westerns using Milo    may complement your Flow Cytometry experiments. Single-cell Western FAQs may be helpful.
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What to consider when high background fluorescence is observed

It is best to use fresh cells or cells that have been fixed for a short period of time to reduce the risk of autofluorescence that contributes to high background fluorescence. It is recommended to run matching unstained cells alongside samples to assess autofluorescence.
Viability dyes (i.e. PI, DAPI,7-AAD, Annexin V, pSIVA) are highly recommended to account for non-specific binding. Tissue dissociation and digestion often results in cell death and high background fluorescence; thus, it is important to discern between viable and dead cells during analysis.

Annexin V/PI Staining Guidelines:
  • Annexin V negative, PI negative = viable cells
  • Annexin V positive, PI negative= early apoptosis
  • Annexin V positive, PI positive = late apoptosis, post-apoptotic necrosis
Increase the buffer volume, number and/or duration of washes, particularly if high background is observed when using unconjugated primary antibodies. Alternatively, the antibody titer might be too high so further antibody dilution may be required.
If you experience high background staining, it may be due to the Fc region of the antibody binding to Fc-receptors rather than antigen-specific binding of the antibody’s Fab region. This is generally unwanted and can be avoided through use of Fc receptor blocking reagents. If high background exists even with use of Fc receptor blockers, consider increasing the concentration or exposure time of such reagents.
Using detergents can result in high background staining. If needed, alcohol permeabilization is a good alternative approach for intracellular targets.
High background can result from poor compensation. Verify that all compensation controls are brighter than the corresponding signal in the experimental sample and prepare N-by-N plots to make sure that the sample has been appropriately compensated.
High background can be due to spillover spreading, which is caused by measurement errors from multiple fluorochromes spilling into each detector and manifests as a spreading of a nominally negative population. This can significantly affect the detection sensitivity and is particularly problematic for the detection of dim markers. If this is the case, our Multicolor Panel Builder can help you adjust your panel accordingly.
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How to choose an optimal antibody for target of interest

Due to their size and inability to efficiently cross the plasma membrane, tandem dyes are only recommended for extracellular staining. Tandem dyes are also referred to as FRET (fluorescence resonance energy transfer) dyes because the emission of the donor fluorochrome (e.g. PE) excites the acceptor fluorochrome (e.g. Cy7).
Conjugated antibodies are recommended for direct labeling of antigens, instead of using pairs of primary and secondary antibodies. Common buffer additives can interfere with the conjugation reaction and limits efficiency. Custom conjugated antibodies with BSA and azide-free packaging may be required. When using conjugated antibodies, the fluorochrome-to-protein (F:P) ratio    for degree of labeling should be calculated for the fluorochrome and target protein.
Indirect detection is more sensitive and vital for effective identification of low abundance antigens and rare epitopes. If you do not get a signal after using an unconjugated primary antibody, check that the correct species of secondary antibody was used for detection.
With indirect detection, cross-species reactivity of secondary antibodies is often a problem. Antibody labeling kits can eliminate the need for the use of secondary antibodies to reduce the number of incubation and wash steps while simultaneously eliminating background due to cross-species reactivity.
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When to use fixation and permeabilization during sample preparation

It is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location.
When both intracellular and extracellular staining are to be performed, it is advised to perform extracellular surface staining first, as the reagents used for fixation and permeabilization tend to decrease surface antigen availability.
Cell fixation: Fixation and permeabilization protocols should be optimized to prevent cell lysis. When fixing cells, it is recommended to follow manufacturer instructions for fixation buffers. If no instructions are given, fixation should not exceed 30 minutes. Certain proteins can be more sensitive to fixation than others such that fixing cells with 4% formaldehyde can result in diminished fluorescence signal. In these cases, 0.5-1% formaldehyde may be more appropriate.
Permeabilization Buffers for intracellular targets: Due to a cell’s ability to regenerate its membrane, cells must be kept in permeabilization buffer to prevent the loss of membrane permeabilization while staining.
  • When assessing antigens close to the plasma membrane or soluble cytoplasmic antigens, mild cell permeabilization without fixation may be required.
  • When assessing intracellular targets using detergents, 0.1-0.5% of Saponin, Triton® X-100 or Tween® 20 in PBS will mildly permeate the cellular membrane instead of complete dissolution of the membrane.
  • More vigorous detergents such as Triton or NP-40 at a concentration of 0.1–1% in wash buffer is a suitable permeabilization buffer for nuclear antigen staining. These detergents dissolve the nuclear membrane completely, allowing antibodies to interact with nuclear envelope and nuclear matrix targets.
  • Organic solvents (i.e. methanol/acetone) are the most vigorous surfactants used to permeabilize membranes by dissolving membrane lipids for intracellular staining. The intracellular flow cytometry protocol using alcohol is often required when detergents produce high background staining. It is important to note that methanol may decrease PE and APC conjugate signals so alcohols should be avoided when possible. However, Alexa Fluor® and Janelia Fluor® conjugated antibodies are compatible with alcohol permeabilization.
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How to optimize separation between wavelengths

Novus provides a multitude of tools to help design and troubleshoot flow cytometry experiments.

Spectra Viewer
  • Select lasers and add filters to determine if an instrument configuration will appropriately excite and detect the fluorescent probe of interest.
  • Add multiple fluorescent probes to determine if they are compatible when multiplexing.
  • Minimize spillover spreading by selecting fluorescent probes that do not have overlapping emission spectra. This avoids reducing the detection sensitivity.
Multicolor Panel Builder
  • Simply choose your pre-configured instrument, add your markers, and quickly search for validated antibodies that are compatible with your instrument.
  • Visualize the spectra of individual fluorochromes to assess spillover. Learn more about panel design.
  • With the built-in Antigen Density Selector tool, your target is assigned to the appropriate fluorochrome based on antigen density expression. For highly expressed antigens, conjugated fluorochromes with lower intensities are recommended. Use the brightest fluorochrome for the lowest expressing antigen in your panel.

View An Introduction to Successful Flow Cytometry Panel Design    webinar for more in-depth information.

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How to best compensate for data analysis

Compensation relies on an accurate calculation of median fluorescence intensity and if there are too few events, this will not be calculated accurately. Thus, for single-stained compensation controls the number of positive events collected should be above 5,000.
Flow cytometry is crucially dependent on the inclusion of appropriate controls, such as FMO (fluorescence-minus-one) and isotype controls, which enable spectral overlap and non-specific binding of reagents to be evaluated and considered. It is critical to use single-color controls for compensation when gating.
Single stained samples (single-color controls) are needed for each fluorochrome used in the experiment. Spectral overlap is assessed between different fluorophores and adjusted for algorithmically prior to data analysis. These can be set up using cells or compensation beads (also known as antibody capture beads). A matched unstained sample needs to be included, i.e. unstained cells for compensation controls using cells and unstained beads for compensation controls using beads.
Instrument controls such as calibration beads are required to determine and optimize performance of the flow cytometer.
Fluorescence-minus-one (FMO) controls are useful for interpreting flow cytometry data and for setting gates. Preparing FMO controls involves staining cells with all reagents except one. Assuming appropriate titration of all reagents, FMO controls provide a reliable assessment of background in a multicolor experiment. FMO controls allow for accurate discrimination between negative and positive cell populations, particularly if cell populations are rare or not clearly defined.
Isotype controls help determine the potential contribution of non-specific background staining, reducing the risk of false-positive results. It is recommended that isotype controls be prepared alongside the conjugated primary antibody to ensure consistent labeling. Used in place of the primary antibody, isotype controls serve as a negative control. This is only meaningful if the isotype control is of the same species and isotype, is conjugated to the same fluorochrome (in the case of tandem dyes, to the same tandem lot), and conjugated at the same F:P ratio   .
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How to read flow cytometry and interpret results

What is flow cytometry? Fluorescent dyes accept light energy at a given wavelength (excitation) and re-emit at a longer wavelength (emission). Linking such fluorochromes to an antibody or using fluorescent molecules that bind to cellular components allows identification of protein expression profiles of select cellular populations by flow cytometry. Go in-depth to learn how flow cytometry works.
Forward and side scatter allows us to separate intact cells from debris, as well as discern major cellular subsets (e.g. of the immune system) according to physical properties.
Do you have high side scatter with low forward scatter? This may indicate the cells are lysed. Assess cell viability and amend experimental protocol to reduce vortexing after permeabilization.
Are your samples poorly fixed or permeabilized? Experimental optimization is suggested to avoid cell lysis caused by high centrifugation speeds and vortexing.
Are your samples poorly fixed or permeabilized? Experimental optimization is suggested to avoid cell lysis via high centrifugation speeds and vortexing.
It is important to consider both the proportion of cells and the intensity of the fluorochrome of interest. The fluorescence intensity for each parameter and gene expression patterns between samples can be calculated by the relative expression of antigens in a population of cells identified using forward and side scatter plots. It is important to set your initial gate using the antigen of interest in the correct cell population.
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Different types of cytometry

Before starting your experiments, consider what type of cytometry is best suited to your scientific question and potential down-stream procedures.
Conventional flow cytometry uses dedicated optical filters to identify distinct fluorochromes for intracellular and extracellular targets. Modern instruments can theoretically detect up to 50 parameters, and manufacturers are working on expanding the repertoire of optically distinct fluorochromes to make this a reality.
Spectral cytometers disperse fluorescence signals across a detector array to analyze the full emission spectrum of each fluorochrome across all wavelengths rather than using specific bandpass filters. Thus, the main difference to a conventional flow cytometer is in the collection of light. Spectral cytometry eliminates the need for dedicated optical filters for each fluorochrome, providing more flexibility in the types and combinations of fluorescent probes being assessed.
Imaging flow cytometry can be used to assess cellular localization of a specific probe, evaluate cell-cell interactions, perform morphological analyses or spot counting. This type of flow cytometry uses objective lenses to capture images of the cells at the interrogation point and relies on charge-coupled device (CCD) cameras to capture fluorescence at multiple wavelengths.
Mass spectometry, such as CyTOF, is a variation of flow cytometry in which heavy metals instead of fluorochromes are conjugated to antibodies. The unique mass signatures of these stable metal isotopes are detected by time-of-flight mass spectrometry and enable identification of over 40 cellular targets on a single cell. While the other types of cytometry technologies measure properties of intact cells, CyTOF vaporizes the cells prior to measurement, making it impossible to sort cells for downstream assays. The advantage of CyTOF over fluorescence cytometry is due to the nature of the read-out, there is no need for compensation. Lightning-Link® Metal Labeling Kits allow isotopically pure metal conjugations to be set up in less than 30 seconds, simply by adding a solution of the antibody to a lyophilized mixture containing a proprietary activated metal ligand.
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Additional Questions?

Flow Cytometry Handbook »

  

Flow Cytometry Basics for the Non-Expert

 

We would like to acknowledge Dr. Yolanda D. Mahnke for input to our flow cytometry troubleshooting guide. President at FlowKnowHow LLC    , a consulting company focused on flow cytometry, Yolanda is an internationally renowned specialist in flow cytometry with a strong background in cancer and vaccine research. She is a co-initiator of OMIPs, a publication platform in Cytometry Part A for highly optimized multicolor immunofluorescence panels.