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Flow Cytometry Troubleshooting Guide

Problem Possible Causes Recommendation
Problem Possible Causes Recommendation

Weak or no fluorescence signal detected by flow cytometer.

Target expression was insufficiently induced by treatment.

Optimize treatment conditions for successful and measurable induction of each target.

If using PBMCs, avoid frozen samples. Isolate fresh cells whenever possible.

Include the appropriate controls:

  • Unstimulated/untreated control
  • Isotype controls
  • Cells alone (unstained) control
  • Positive control

Inadequate fixation and/or permeabilization of sample.

If the target of interest is intracellular make sure that the appropriate fixation and permeabilization protocol is used. Different forms of fixation and permeabilization may be required, depending on the target and whether surface marker staining is carried out at the same time. Formaldehyde fixation can be used in conjunction with Saponin, Triton X-100 or 90% methanol (ice-cold), to allow for permeabilization. 70% ethanol (ice-cold) can sometimes be used as an alternative method of fixation, particularly when performing cell cycle analysis.

Note that fixation can sometimes compromise the detection of certain surface epitopes. Thus it is recommended to test how the extracellular epitope of interest responds to the fixative in question prior to proceeding with dual extra- and intracellular staining.

Fixative should be added immediately after treatment and should be at a high enough concentration to inhibit any phosphatase activity (CST recommends 4% formaldehyde).

Ensure that methanol-free formaldehyde is used to prevent the loss of intracellular proteins, which may occur due to cell permeabilization before sufficient cross-linking is achieved.

For methanol permeabilization, it is critical to chill cells on ice prior to drop-wise addition of ice-cold methanol, to prevent hypotonic shock.

A weakly expressed target was paired with a dim fluorochrome.

Always use the brightest fluorochrome (e.g. PE) conjugate to detect the lowest density target (e.g. CD25) and the dimmest fluorochrome (e.g. FITC) conjugate to detect the highest density target (e.g. CD8).

Note that some fluorochromes are more amenable for use in intracellular staining than others. Factors to consider are the size, conformation and stability of the fluorochrome. For instance, fluorochromes with a larger molecular weight e.g., certain synthetic dyes, tend to be unfavorable for intracellular, particularly nuclear, staining due to a difficulty in efficiently penetrating through the cell and nuclear membranes.

If a secondary antibody was used for detection, the specificity of the secondary antibody may be incorrect (e.g. rabbit IgG primary antibody incorrectly detected with an anti-mouse IgG PE conjugated secondary Ab).

Ensure that the secondary antibody was added at the recommended concentration and that it is against the correct host species of the primary antibody.

The laser and PMT settings on the flow cytometer may not be compatible with the fluorochrome that is conjugated to the primary or secondary antibody.

Ensure that the laser wavelength and PMT settings match the excitation and emission wavelengths of the fluorochromes being used.

Scatter properties of cells are suboptimal (e.g. low Forward and Side Scatter values).

Incorrect instrument settings.

Ensure that proper instrument settings are loaded into the cytometer either by using a control sample or by using instrument settings from a previous experiment.

Poorly fixed/permeabilized sample prep.

Follow the CST standard Flow protocol for cells or the Flow Alternate protocol for human whole blood. It is important that the permeabilization step with 90% methanol be done as instructed i.e., introduce 90% ice-cold methanol drop-wise onto your cell pellet, while gently vortexing to ensure homogeneous permeabilization and to avoid damaging the cells by rapid immersion into a hypotonic solution such as methanol.

Flow cell of cytometer may be clogged.

Unclog cytometer as per manufacturer's instructions (typically run 10% bleach for 5-10 min, followed by dH2O for 5-10 min).

When running samples to examine cell cycle distribution the histogram for DNA content does not resolve the distinct phases of the cell cycle, i.e., G0/G1, S and G2/M phases.

Incorrect flow rate.

Ensure that your samples are being run at the lowest flow rate setting on your cytometer. High flow rates will give rise to high coefficients of variation (CVs), leading to a loss of resolution of the different phases of the cell cycle.

Insufficient staining with Propidium Iodide/RNase (PI) solution.

Resuspend cell pellet directly in PI/RNase solution (#4087) and incubate for at least 10 min; if PI cannot be used due to fluorescent channel configurations, an alternate DNA dye such as DRAQ5 (#4084) or DAPI (#4083) can be used instead.

Cells are not proliferating.

Cells should be harvested during asynchronous and exponential growth to ensure that all phases of the cell cycle are accurately represented.

High signal in negative cell populations.

Off-target cell populations (e.g., monocytes) may express Fc surface receptors, which may bind the Fc portion of the primary or secondary antibody, causing non-specific staining.

Try blocking cells with Bovine Serum Albumin, Fc receptor blocking reagents or normal serum from the same host as the primary and/or secondary antibody prior to staining.

Use a control containing only the secondary antibody to determine if background staining is originating from the primary or secondary antibody.

Perform additional wash steps between antibody incubations.

Incomplete red blood cells lysis when using the Flow Alternate Protocol.

Additional washes may be necessary to eliminate red blood cell debris.

Add up to 3 washes between each step, if necessary.

The 0.1% Triton X-100 solution may not be fresh.

Use fresh reagents and closely follow the CST Flow protocol, which has been extensively optimized.

The Flow Alternate Protocol is derived from a publication by Chow et al., (2005) Cytometry Part A 67A:4–17, which can be used as a further resource.

Antibody works for other applications but not for flow cytometry.

Antibody is not recommended for flow on product data sheet.

The antibody was either not tested or did not pass the criteria required by CST for flow validation.

Antibodies approved for immunofluorescence only may be tested for flow cytometry by performing a titration series to determine the optimal concentration.

Contact technical support for further information regarding testing status/history of the antibody.

High background.

Too much antibody.

Use the recommended antibody dilution. CST recommended dilutions have been optimized for 105-106 cells. When using low cell numbers, we recommend performing your own titration.

Two-step antibody staining.

If possible, avoid the use of biotinylated antibodies. These tend to cause high background levels when performing intracellular staining due to the detection of naturally occurring endogenous biotin within the cell by streptavidin or anti-biotin secondary antibody conjugates.

Whenever possible, perform direct staining.

Presence of dead cells.

Use a viability dye such as PI or 7-AAD to gate out dead cells when performing live cell surface staining. However, when staining is to be done on fixed cells, use fixable viability dyes, e.g., eFluor, which are designed to withstand fixation and can be used in conjunction with intracellular staining.

High autofluorescence.

It is noteworthy that certain cell types (e.g., neutrophils) naturally exhibit higher levels of autofluorescence as compared to others.

Two non-mutually exclusive approaches can be taken to address this issue:

  1. Use fluorochromes that emit in red-shifted channels, in which autofluorescence is minimal. For instance, APC is far less conducive to autofluorescence when compared to FITC or Pacific Blue.
  2. Use very bright fluorochromes in the channels that do exhibit autofluorescence. This means using Alexa Fluor 488 or Brilliant Blue 515 instead of FITC, Brilliant Violet 421 instead of Pacific Blue, and favoring biotin-streptavidin and primary-secondary steps to amplify signal.