How many cells do I need to stain?

Adopted from: https://voices.uchicago.edu/ucflow/2020/01/16/how-many-cells-should-i-stain-the-impact-of-cell-concentration-and-antibody-concentration/

One common question we ask ourselves when starting flow cytometry is: how many cells should I stain? Keep in mind that when you digest an organ/tumor or take blood out of the animal, it would be ideal to run the entire population of cells as this is not impossible. Ideally, we would need both time and money to run 100 million cells, but it is not usually feasible. The work around is to take a sample of your original sample and hope you have obtained a representative sample for the analysis. This is why technical replicates and biological replicates improve rigor to your data.

Do you follow the default event number on the instrument? Cytek, for example, will default to 5000. This may be enough for homogenous sample (in vitro cell culture or beads), but not statistically sound for heterogenous samples such as single cell suspension prepared from an experiment.

Ultimately, the number will depend on how you answer the following questions:

1. What are my experimental goals? Endpoints? What am I comparing?  
2. Is my population of interest rare? What is overall percentage of the cell that I’m interested in?

For example, you are looking to measure regulatory T cells from spleen or tumor sample, they can make up to 1% (blood, tumor, etc). Analyzing 100,000 cells total, Tregs would make up 1000 events. Would that be enough? It is recommended to have at least 100 – 500 events in the rarest population so collecting 100K would be barely enough.

Before deciding on the number of cells though, you need to first titrate your antibody.

Do this in 2 ways:

1. Keep the number of cells the same, test a variety of different antibody concentration.
2. Keep the concentration of antibody the same but vary the number.
3. What is your outcome?

At the end of this test, you will need to keep the number of cells and the concentration the same for your subsequent experiments. Same number of cells should be used for your experiment and preparation of FMOs.

Why titrate? Titration will determine the SI – the optimal antibody concentration for a given number of cells.

The important thing is to keep the antibody concentration exactly the same and then keep the cell concentration similar between the controls and samples, knowing that a range of cell concentrations can work for a single antibody concentration. Keep things consistent, including incubation time.  Use the same reagent, protocol, and instrument to obtain reproducible results.

Some physics: what motivates binding of the antibody to its target? Consider the following scenario: you have 1 million cells and 0.1mg/mL antibody concentration in either 200 uL or 500 uL volume. Antibody might be able to find its target faster in 200 uL because of the proximity between itself and the target (i.e. Law of Mass Action).

Another thing to consider is affinity and avidity. These two factors will dictate how readily the antibody binds to its target (speed and tightness of binding).