Flow Cytometry Troubleshooting

Little or no signal may be due to: (1) the primary antibody concentration, (2) the cell collection method, or (3) the flow cytometry assay itself.

• Please check that your secondary antibody recognizes the primary antibody: most of our antibodies are generated in rabbits.
• If you have previously used your secondary antibody with successful results, we suggest trying longer incubation times with the primary antibody (up to 2 hours at 4°C) in a smaller reaction volume.
• Your primary antibody concentration may be too low. Try using more primary antibody or set up a series of dilutions to determine the optimal concentration for your system.
• If you are trying to stain an intracellular protein, you’ll need to permeabilize your cells first.
• If you are attempting to detect cell surface proteins, they may have become internalized. Trypsin can induce the internalization of cell surface proteins so you may need to alter the cell detachment method. You can reduce the internalization of cell surface proteins by adding NaN₃ or by keeping your assay reagents on ice.

High background levels are most likely due to too much primary antibody concentration or insufficient blocking. Both of these cases can independently lead to a high signal in what should be negative cell populations.

• If you are using direct flow cytometry, use a suitable conjugated IgG isotype control.
• If you are using indirect flow cytometry, use your secondary antibody alone as a negative control.
• Too much antibody will produce a lot of background. Test different antibody dilutions to determine the optimal concentration.
• Your antibody may be binding off-targets such as the Fc receptors. You can try an Fc receptor blocking reagent to minimize the off-target binding.
• There may be doublets in your cell population, i.e., dividing cells. Try adding EDTA to your buffer or filtering the cells through a 30 µm filter.
• It’s possible that the flow cytometer equipment settings may need adjusting: if the offset is too low, or the gain too high, you will generate background signal.
• If you are not using a conjugated primary antibody, you may need to simply increase the wash times, or add extra wash steps to your protocol.
• When running multiple fluorochromes, spillover can occur. Ensure that you have used a multicolor panel building to confirm that you have a workable combination of fluorochromes.
• When choosing the fluorochromes, the brightest fluorochrome for the target with the lowest expression or density should be selected. Conversely, choose the dimmest fluorochrome for those targets with the highest expression or density.
• If you have high side scatter background it could be due to bacterial contamination, which will autofluoresce and give high event rates.

If your flow cytometer cannot consistently distinguish between individual cells, your event rate will appear abnormal. Remember, you may need to obtain as many as 10⁷ events for significant detection.

• If the event rate is too low, ensure that the cells are properly mixed and that the population is between 1 x 10⁵ and 1 x 10⁶ cells/ml.
• If the event rate is too low, your cells could be clumping together. Make sure you sieve the cells before they are acquired and sorted to remove any debris.