By Ryan Hamnett, PhD
Flow cytometry uses light to characterize and measure heterogenous suspensions of cells based on their physical characteristics and fluorescence. Flow cytometry typically uses antibodies conjugated to fluorophores to target extracellular markers in order to define cell populations. Our troubleshooting guide will help to solve any issues that might be encountered with a flow cytometry experiment.
| Potential issue: | Possible solution: |
|---|---|
| Antigen target not present | Check literature to confirm that target is expected in cell type Check whether stimulation is required for expression of target |
| Target is intracellular | Fixation and permeabilization are necessary for intracellular antigens Perform procedure on ice to prevent internalization of cell surface proteins |
| Intracellular target inaccessible | Ensure adequate permeabilization Try an alternate permeabilization method if possible Trypsin treatment of some cell lines results in internalization of cell surface proteins, so alternative detachment methods may be needed Fluorophore-antibody conjugate may be too large, so try with a lower molecular weight fluorophore |
| Target has been secreted | Treat with brefeldin A or monensin to prevent secretion of intracellular proteins |
| Inadequate fixation when staining for intracellular antigen | Add fixative immediately after treatment Trial alternative fixatives If using methanol, chill cells beforehand and add methanol drop-wise to prevent hypotonic shock |
| Insufficient antibody | Optimize concentration of antibody |
| Antibody does not recognize antigen in test species | Use an antibody with appropriate reactivity for species under investigation |
| Primary and secondary antibodies are incompatible when performing indirect staining | Use a secondary antibody that will recognize the host species of the primary antibody |
| Antibody is not suitable for flow cytometry | Choose an alternative antibody that has been validated for flow cytometry |
| Fluorophore has faded or bleached | Use fresh fluorophore-antibody conjugate |
| Antibodies stored incorrectly | Aliquot and store antibodies according to manufacturer’s instructions. Some fluorophores, such as PE or APC, should not be frozen |
| Conjugation kit failed | Use a pre-conjugated primary antibody |
| Dye brightness and antigen expression are not well matched | Match the brightest fluorophore with the lowest expressed antigen |
| Lasers not aligned | Run flow beads to check alignment, particularly for steam-in-air cytometers. Alignment should only be performed by adequately trained personnel such as a service engineer |
| Offset too high or gain too low | Establish settings using a positive control sample and optimize offset and gain |
| Incorrect laser or detection settings are being used | Ensure the channel and laser being used correspond to the fluorophore’s excitation and emission spectrum |
| Potential issue: | Possible solution: |
|---|---|
| Antibody is binding to Fc receptors | Block Fc receptors using BSA, serum or Fc blocking reagents Perform extra wash steps Include a secondary antibody only control to determine if off-target staining is due to primary or secondary antibody in indirect staining Include an isotype control |
| High autofluorescence | Check base level of autofluorescence by including an unstained control |
| Presence of dead cells | Exclude dead cells by including a viability dye such as PI or 7-AAD. Note that some viability dyes are incompatible with cell fixation |
| Excess antibody | Optimize antibody concentration Increase stringency of wash buffer by adding mild detergent |
| Gain too high or offset too low | Establish settings using a positive control sample and optimize offset and gain |
| Cross-reactivity between secondary antibody and another primary antibody | Use cross-adsorbed secondary antibodies Use a pre-conjugated primary antibody |
| Potential issue: | Possible solution: |
|---|---|
| Antibody concentration too high | Optimize antibody concentration |
| Excess antibody trapped in cells when staining for intracellular antigens | Include more washing steps and increase stringency of washes by adding a mild detergent to wash buffer |
| Inadequate blocking | Block Fc receptors using BSA, serum or Fc blocking reagents |
| Potential issue: | Possible solution: |
|---|---|
| Blocked or clogged cytometer | Dilute the sample, for example down to 5 x 105 cells/ml Clean the cytometer |
| Cells lost during procedure | Handle cells carefully to prevent cell loss Run 1 x 106 cells/ml |
| Cells are clumping | Use a more aggressive enzyme treatment to dissociate cells Try an alternative fixation approach Exclude cell clumps using a cell strainer Perform procedure on ice to reduce apoptosis or necrosis |
| Potential issue: | Possible solution: |
|---|---|
| High side scatter due to cell lysis | Handle cells carefully, reducing centrifugation speed or vortexing intensity |
| Autofluorescence or high event rate due to bacterial contamination | Ensure sample is not contaminated |
| Multiple populations observed when only one expected | Multiple populations are expressing the antigen and will require an extra marker to distinguish them Cell doublets are present and can be prevented by gentle mixing before staining and running on the cytometer, or filtering with a cell strainer |
| Incorrect cytometer settings | Optimize settings using a positive control sample |
| Dead or degraded cells | Use fresh cells, and run them on the cytometer as soon as possible Fix cells at the end of the staining procedure if they will not be analyzed immediately |
| Certain reagents can affect antigens or antibody staining | EDTA, sodium azide, lysing solutions and fixatives can all affect certain antigens or cell types |
| Potential issue: | Possible solution: |
|---|---|
| Cell viability has changed | Check cell culture conditions to ensure that cell numbers are consistent between runs |
| Cytometer is incorrectly calibrated | Calibrate instrument |
| Different batch of antibody used | Keep antibody batch consistent between experiments if possible, especially for tandem dyes |