I ran some beads and noticed there are two or three populations. I thought the beads were only a single size? What’s going on?
What this user is noticing is likely aggregation of the beads. FSC/SSC (forward scatter / side scatter) dot plots provide general information in terms of size (FSC) and granularity (SSC). When two particles are stuck together, the instrument will detect a larger scatter than a single particle, and thus the FSC/SSC signal will increase – shifting the measured result to the right, and up. This can be seen in the below figures, where our main population of singlets represents the majority of measured particles (~ 77%), but a subset of doublets exists as well. After additional mixing, we were able to increase the singlets to 95%. Aggregation can also be observed in histograms, and will generally present a signal that is a multiple of the original intensity. That is, if you have a forward scatter signal of x for singlets, then doublets will be 2x, and triplets will be 3x (see figure 2).
- Once identified, there are a variety of ways to combat aggregation, either on the analysis end, or the sample handling end.
- Be sure to gently mix your bulk suspension before sampling. This helps ensure the particles are in a monodispersed state. If low levels of aggregates are present, consider tolerating them, you’ll gate them out anyway! Cytometers analyze thousands of events in a typical run, and it’s not unusual for a small % to be present as aggregates. We generally advise users to aim for 90% or more singlets.
- Review your sample handling. We’ve had instances of users centrifuging our beads too hard/long, which makes complete resuspension difficult.
- Attempt greater agitation efforts. It may not need to be much! For instance, we’ve resolved aggregation by simply changing “tapping the tube” to “hard tapping of the tube”. For more serious instances, use pulse vortexing. Be considerate of what product you’re using (protein coated or surface labeled products should be handled with more gentle techniques).
- Add a surfactant. Surfactants go a long way in helping resolve aggregation, and maintaining monodispersity. A little goes a long way, often 0.01 - 0.1% is more than adequate. We include non-ionic surfactant in many of our flow products.