1.     Weak Fluorescence Intensity or No Fluorescent Signal

​

The antibodies are degraded or expired:

• Ensure that antibodies are stored as per the instructions of manufacturer.

• Keep track of antibody stocks; make sure products are not expired.

• If titrating antibodies and storing aliquots of the same, add sodium azide in the storage buffer at 0.09%.

 

The fluorescence of the fluorochrome has faded

• Be sure to store the conjugated antibodies away from light exposure. Fresh antibodies will be needed.

 

The antibody concentration is too low for detection

• Titrate the antibodies before use to find the optimal amount to use for your specific experiment.

• Use negative (unstained) and positive controls.

 

Expression of target antigen is too low

• Check literature for antigen expression in different cell types and use a suitable positive control.

• Whenever possible, use freshly isolated cells as opposed to frozen samples.

• Optimize cell culture/stimulation protocols in case the antigen (e.g. cytokines) expression depends on prior in vitro treatment.

 

Antigen-antibody binding is sub-optimal

• Check the species specificity of the antibody.

• Optimize the antibody incubation time and temperature.

• Consider using biotinylated primary antibodies and an additional biotin-steptavidin step to amplify the signal.

 

The intracellular antigen is not accessible

• Optimize cell permeabilization protocols.

 

The intracellular antigen is getting secreted

• Target must be membrane bound or cytoplasmic to be detected. Try using a Golgi blocker such as Brefeldin A.

 

The surface antigen is getting internalized

• Perform all protocol steps at 4°C and use ice cold reagents. We recommend you permeablize on ice.

 

The fluorescence on stained cells has bleached

• Acquire the cells immediately after staining.

• Add a fixative (like PFA) to the samples if storing for an extended duration; alcohol fixatives are best avoided.

 

A low expressing antigen has been paired with a dim fluorochrome

• Always pair the weak antigens with bright fluorochromes such as PE or APC.

 

The primary and secondary antibodies are not compatible

• Use a secondary antibody that was raised against the species in which the primary antibody was raised.

 

The laser and PMT settings are not compatible with fluorochrome or PMT voltage is too low for the fluorescent specific channel

• Ensure that proper instrument settings are loaded prior to acquisition.

• Use suitable positive and negative controls to optimize settings for every fluorochrome.

 

The fluorescent signal is over compensated

• Use MFI alignment instead of visual comparison to compensate.

 

2.     Saturated or Excess Fluorescent Signal

​

The antibody concentration is too high

• Titrate the antibodies before use to find the optimal amount to use for your specific experiment.

• Use appropriate positive and negative controls.

​

Unbound antibodies are trapped in the cells in the case of intracellular staining

• Wash the cells adequately after each antibody incubation step and include Tween or Triton X in wash buffers.

​

A high expressing antigen is paired with bright fluorochrome

• Always pair strong antigens with dimmer fluorochromes such as FITC or Pacific Blue.

​

The PMT voltage is too high for the fluorescent specific channel

• Ensure proper instrument settings are loaded prior to acquisition.

• Use suitable positive and negative controls to optimize settings for every fluorochrome.

​

The fluorescent signal is under-compensated

• Use MFI alignment instead of visual comparison to compensate.

​

Inadequate blocking

• Add 1% to 3% blocking agent with antibody as well as a blocking step (dilute antibodies in blocking solution), and increase the blocking time.

 

3.     High Background or Non-specific Staining

 

​

Excess, unbound antibodies are present in the sample

• Wash cells adequately after every antibody incubation step

​

Non-specific cells are targeted

• Include an isotype control to subtract any Fc binding signal.

• Block the Fc receptors on cells with Fc blockers, BSA or FBS prior to antibody incubation.

• Include additional washing steps.

• Include a secondary antibody-conjugate control to subtract any non-specific signal from the conjugate.

• Select a secondary antibody that does not cross react.

​

High auto-fluorescence

• Always include an unstained control to subtract the auto-fluorescence signal.

• For cells with naturally high auto-fluorescence (e.g. neutrophils), use fluorochromes that emit in the red channel where auto-fluorescence is minimal (e.g. APC).

• If the above solution is not possible, use very bright fluorochromes for these cell types to amplify the signal above the auto-fluorescence level.

• Avoid storing the cells in a fixative solution for long durations; analyse cells soon after staining if possible.

​

Presence of dead cells

• Always sieve the cells once before acquiring and sorting to remove any dead cell debris.

• Include viability dyes like PI or 7-AAD to gate out any dead cells.

• Use freshly isolated cells as opposed to frozen cells whenever possible.

 

4.     High Background Scatter or Abnormal Scatter Profile of Cells

​

The cells are lysed or damaged

• Optimize sample preparation to avoid cell lysis.

• If possible, do not vortex or centrifuge cells at high speeds.

• Use fresh buffers.

• Avoid storing the stained cells for long durations.

​

Bacterial contamination

• Store stained cells and antibodies properly to avoid bacterial growth. Bacteria will exhibit low levels of auto fluorescence.

• Practice proper sterile cell culture techniques to prevent bacterial contamination.

​

Incorrect instrument settings for scatter

• Ensure proper instrument settings are loaded prior to acquisition.

• Use fresh, healthy cells to correctly set the FSC and SSC settings for that cell type.

​

Presence of dead cells

• Always sieve the cells once before acquiring and sorting to remove any dead cell debris.

• Use freshly isolated cells as opposed to frozen cells whenever possible.

​

Presence of un-lysed RBCs (red blood cells)

• Ensure RBC cells lysis is complete – check using a microscope.

• Use fresh RBC lysis buffer.

• Wash as many times as needed to remove RBC debris.

• If using PBMCs after a ficoll gradient, optimize the procedure to minimize RBC contamination in the lymphocyte interphase.

 

5.     Abnormal Event Rate

 

​

Event rate is low due to low cell number

• Keep the minimum cell count at 1X106/ml.

• Ensure the cells are mixed well with gentle pipetting.

​

Event rate is low due to sample clumping

• Always sieve the cells once prior to acquiring and sorting to remove debris.

• Ensure the cells are mixed well with gentle pipetting before staining, and again before running your samples.

​

Incorrect instrument settings

• Adjust the threshold parameter as needed.

​

No events due to a clogged sample injection tube

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

​

Event rate is too high due to concentrated sample

• Dilute the cell count to 1X106/ml.

​

Event rate is too high due to air in flow cells and/or sheath filter

• Refer to the instrument manual for the appropriate steps.

 

6.     Loss of Epitope

​

Excessive paraformaldehyde

• Paraformaldehye can release methanol in its breakdown, which may affect the staining. Make sure to use only 1% paraformaldehyde.

​

Sample was not kept on ice

• Keep antibodies at 4 °C to prevent loss of activity. This also prevents active phosphatases and proteases from altering the epitope of interest.

​

Sample fixed too long

• Optimize fixation protocol. Fixing the samples for too long may cause damage to cells. Most cells only need to be fixed for less than 15 minutes.