FLOW CYTOMETRY TIPS
We are dedicated to helping our customers achieve exceptional results. While the information below can never be an exclusive solution to every problem you may encounter; it is our hope that you will find the information useful and even beneficial in troubleshooting any problems you may have. Of course, if you find you are still having problems you may submit them to us using this Flow Cytometry Troubleshooting Form.
> No Signal / weak fluorescence
> High fluorescence
> Two cell populations
> High side scatter background
> Low event rate
> High event rate
> Unusual scatter profiles
> Unexpected staining
> Loss of epitope
> High background/high percentage of cells
> No events
NO SIGNAL / WEAK FLUORESCENCE
Signal not correctly compensatedMake sure everything is gated properly by checking the positive single color control and adjust compensation accordingly.
Insufficient antibodyIncrease the antibody concentration to all experimental tubes.
Intracellular target not availableCheck to see if the target protein is intracellular. If so, make sure to permeablize on ice and with cold reagents in order to stop all reactions.
Excess cell numberDilute sample tubes in PBS for recommended concentration
Lasers not alignedRun setup beads and adjust alignment if necessary.
Soluble/secreted target protein Target protein must be membrane bound or cytoplasmic to be detected. A Golgi-block step, such as Brefaldin A, may improve signal.
Offset too high/gain too lowMake sure your positive control has a peak at 102. Check fluorescent signal and adjust voltage (within reason).
Primary and secondary are not compatibleSecondary should be raised against the host species of the primary.
PE antibody doesn't stain but FITC gives good results Paraformaldehye can release methanol in its breakdown, which will affect the staining.
Fluorochrome fluorescence faded Sample may have been left out too long or been exposed too long. Experiment should be repeated with fresh antibody.
HIGH FLUORESCENCE INTENSITY
Antibody concentrationToo much antibody will give high non-specific binding or very high intensity fluorescence.
Excess antibody trapped Ensure adequate washing steps and include tween or triton in the wash buffers. Fluorochrome molecules can be trapped while staining intracellular.
Inadequate blockingDilute antibodies in blocking solution, and increase the blocking time.
TWO CELL POPULATIONS
More than one cell population present expressing target proteinLessen the This may be in part to debris or adequate cell separation. Check expected expression levels from cell types contained in the sample.
Cell doublets presentCells can be filter or sieved to remove unnecessary clumps. Mix cells gently with a pipette before staining and again before acquiring.
HIGH SIDE SCATTER BACKGROUND
Cells lysedSamples need to be fresh and prepared correctly. Do not vortex too violently, or centrifuge too fast.
Bacterial contamination Bacterial will auto fluoresce at low level. This will give a high event rate
Low number of cells/mlOptimal cell conentration for each sample should be around 1x10^6 cells/ml. Ensure cells are gently mixed.
Cells clumped/blocking tubingPipet gently before staining and again before running. It's recommended to use to a filtered tube when running sample.
High number of cellsEither dilute sample to 1 millions cells/mL or adjust the machine setting to low speed.
UNUSUAL SCATTER PROFILES
Showing dead cellsThis may be debris, and gates need to be adjusted. Or all cells could have died during mixing or additonal chemicals added. Repeat experiment with fresh cells.
Activation methods may affect scatter characteristics of cellsThis should be ok if the positive and negative control tubes were properly set up. Or if required, consult a different activation method.
Affect antigen detectionPermablization is required. However, reagents can affect certain antigens, i.e. lysis buffers and EDTA affects platelet markers.
Presence of dead cellsAlways 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.
LOSS OF EPITOPE
Too much paraformaldehydeMake sure to only use 1% paraformaldehyde.
Sample fixed too longFollow proper instructions, most cells only need to be fix for less than 15 minutes. It is recommended to not fix for too long, as this will damage cells.
Sample wasn't kept coldFollow proper protocol instructions, however most antibodies need to be kept at 4C to prevent loss of activity. Also, this prevents active phosphatases and proteases from altering the epitope of interest.
HIGH BACKGROUND/HIGH PERCENTAGE OF CELLS
Gain set too high/offset too lowUse the positive control to set up the flow cytometer correctly again, using the offset to reduce background from small particles and reduce the gain to decrease the signal.
Excess antibodyDecrease the antibody concentration. Detergent can also be added to the wash buffers to ensure any excess antibody is washed away.
Clogged sample tubeUnclog 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).