How Many Cells to Collect for Flow Cytometry
Author: Mike Blundell | Reviewer: Chloe Fenton
To obtain statistically meaningful results in flow cytometry, the number of cells you need to collect depends primarily on the frequency of your target population. Rare populations require significantly higher total event counts compared to abundant populations.
Factors That Influence Cell Collection in Flow Cytometry
The number of cells you need to collect during analysis to have statistically significant results can vastly differ depending on the sample and frequency of your cells. If you have a sample with an abundant cell type, such as T cells in human peripheral blood, which represent around 20% of the total mononuclear cells, you will have to collect and stain less cells than if you are looking at NK cells, which have a frequency of around 5%.
The table below shows an example of how the frequency of cells can affect the number of cells collected.
Table 5. Cell frequency.
Starting Population |
Frequency |
Number Collected |
|---|---|---|
| 1,000,000 | 10% | 100,000 |
| 1,000,000 | 1% | 10,000 |
| 1,000,000 | 0.1% | 1,000 |
How to Interpret the Cell Frequency Table
The table demonstrates the relationship between the frequency of a target cell population and the number of cells that must be collected to obtain meaningful data.
- Higher-frequency populations (e.g., 10%): a larger proportion of the total sample consists of the target cells. This means fewer total events are required to obtain a sufficient number of target cells (e.g., 100,000 collected from 1,000,000 starting cells)
- Medium-frequency populations (e.g., 1%): as the target population becomes less abundant, the number of detected target cells decreases proportionally (e.g., 10,000 collected), requiring careful consideration of total event counts
- Low-frequency or rare populations (e.g., 0.1%): very few target cells will be present (e.g., 1,000 collected), meaning significantly more total cells may need to be acquired to ensure statistical confidence in downstream analysis
In practice, this means that experiments targeting rare populations require acquiring a much larger total number of events to ensure that enough target cells are captured for reliable interpretation.
In addition to the number of cells, the number of markers simultaneously detected to look at cell subsets can affect the number of cells that are needed to be acquired; generally an increase in markers requires more cells.
Finally, performing the right a controls to determine the variation and allow definition a positive or negative is also very important.
More detailed information on collecting enough events can be found in an article by M Roederer in Cytometry Part A. (Roederer M (2008). How many events is enough? Are you positive? Cytometry, 73A:384-385).
Frequently Asked Questions
How many cells should I collect for flow cytometry?
The number of cells required depends on the frequency of your target population. Higher-frequency populations require fewer total events, while rare populations require significantly more cells to be collected to achieve statistically meaningful results.
Why do rare cell populations require more cell collection?
Rare populations represent a very small proportion of the total sample. As a result, more total cells must be acquired to ensure that enough target cells are detected for accurate analysis.
Does the number of markers affect how many cells I need?
Yes. Increasing the number of markers used to define subsets can increase the total number of cells required, as more complex analyses typically require larger datasets to resolve populations accurately.
Are controls important when determining how many cells to collect?
Yes. Controls are essential for determining variation and defining positive and negative populations, and they should be considered when planning how many cells to acquire.
What happens if I collect too few cells?
Collecting too few cells may result in insufficient representation of the target population, particularly for rare cells, leading to reduced confidence in the results and potentially incorrect conclusions.
How does cell frequency affect flow cytometry results?
Cell frequency determines how many target cells are present in a sample. Lower-frequency populations require higher total event counts to ensure accurate detection and statistical confidence.
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Resources
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