Flow Cytometry Troubleshooting Guide

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

Include the appropriate controls:

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.

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).

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.

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.

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.

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.

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.