Richard Cuthbert
Counting is something we are all familiar with but it’s not always as simple as one, two, three. Measuring the concentration of cells in suspension is a common but surprisingly complex task in cell biology that is easy to get wrong. This article explores ten factors that can influence cell counting, gives advice on how to improve accuracy, and dispels some commonly held myths.
We typically first learn to count between the ages of two and four. It’s such a fundamental part of our cognitive make up that if someone were to call into question our ability to count accurately, we may feel insulted. However, a task involving counting is not always as straightforward as it may initially seem. For example, dyslexia can affect a child’s capability for simple counting, but student performance often improves with strategies such as pattern recognition. Similarly, when it comes to counting cells in suspension, there are many complicating factors that affect accuracy, which can be improved with the right strategies. Here, we detail some of those complications, suggest approaches to overcome them, and dispel some common myths associated with counting cells.
1
Sample preparation
How you handle your cells is crucial both in terms of the accuracy of your cell counts and how your cells behave post-counting. Here are some general points to remember:
- Aim to passage adherent cells at 70–80% confluency. This will maximize cell viability, maintain steady cell growth, and reduce trypsinization time
- Optimize digestion protocols for solid tissues. Insufficient digestion will result in reduced numbers of liberated cells. Excessive digestion can significantly impact cell viability
- Always wash your cells prior to counting (typically using phosphate buffered saline). This will reduce the amount of debris in your cell suspension
- Using an appropriate seeding concentration after counting ensures optimal viability in your subculture
2
Adequate cell suspension (or sample) mixing
There are many methods used to mix samples, each with advantages and drawbacks:
- Pipetting the cell suspension up and down is often thought of as the gentlest method for most samples. However, pipetting tends to cause shear stress, especially in viscous samples. Pipetting a significantly smaller or larger volume than the sample itself can be ineffective or introduce bubbles, respectively
- Vortexing is effective, but take care if using unsealed containers such as tissue culture plates; spilling can lead to cross-contamination and loss of sterility
- Flicking or inverting a sealed vessel is convenient and quick, but not as effective as vortexing
3
Where and when to count
Once your sample is mixed, it’s important to count as soon as possible. Pipet from the middle of the sample and not the top or bottom of the sampling vessel. Cells settle to the bottom surprisingly quickly, and different cell types and sizes settle at different rates. Therefore, the top of the sample will be slightly diluted, the bottom will be more concentrated, and the middle will be the most representative area.
4
Avoid aggregates or clumping
Aggregates in your suspension represent a significant potential source of error. Aggregation can occur due to the release of DNA from dead cells. This is a particular problem when defrosting cells after cryogenic storage.
- If you have a plentiful supply of cells, filter with a 30 µm cell strainer to remove any aggregates
- If you have a limited supply of cells, consider incubating your sample in a DNase solution to break up aggregates prior to filtering
5
Choose the correct dilution for counting
It’s important to work at a dilution at which your counting equipment is designed to work well. When using manual cell counting equipment, counting more concentrated samples will improve accuracy but will also take longer. An automated cell counter largely removes the need for dilution since they often work effectively at a broad range of dilutions.
- When using a hemocytometer, aim for a dilution that allows you to count at least 100 cells
- When using an automated cell counter consult your instrument’s user guide for the optimal concentration range to provide the most reproducible data. Be aware that when the sample concentration is close to the lower and upper limits of the optimal concentration range, cell counts will be less accurate
- In cases where the concentration of cells is low and cannot be easily increased, manual methods may prove more accurate than automated counting
6
Perform a viable count
The viable cell count is possibly more important than the total count. Only viable cells should be considered when reseeding, and monitoring the number of dead cells helps to assess your upstream sample preparation. Uptake of trypan blue solution is an efficient method to highlight non-viable cells. However, an old trypan blue solution can form precipitates and make accurate counting difficult or even impossible.
7
Minimize debris
Debris can be difficult to distinguish from cells, leading to inaccurate counts. While difficult to avoid completely, adequate sample washing and filtering of any buffers used for cell resuspension or dilution can help. If the amount of debris in your cell preparation is severe, consider using density gradient centrifugation to remove debris from your sample.
8
Standardize
Small changes can have big consequences, from the way you choose to mix your samples to the pipets and even the type of plasticware you use. The more rigidly you standardize your sample preparation processes and how you perform your sampling and counting, the more reproducible your results will be.
9
Perform replicates
As with any measurement, there is an inherent margin of error regardless of how rigorous your methodology is. Averaging across replicates increases the precision of your measurements.
10
Consider an automated cell counter
It takes experience to count cells accurately using a hemocytometer. However, even experienced users may not agree when differentiating cells from debris or viable cells from nonviable cells. Additionally, even with practice, it takes approximately four minutes to count cells manually. A protocol that requires three counts per sample performed on five samples equates to one full hour of cell counting. In contrast, automated counters take only seconds per sample.
Bio-Rad’s TC20 Automated Cell Counter. The TC20 Cell Counter enables accurate cell counting in less than 30 seconds.
Table 1. Considerations when selecting an automated versus manual counting method. User preferences can often be influenced by the balance between cost, convenience, contamination, efficiency, and accuracy.
| Automated Cell Counter | Manual Cell Counter (Hemocytometer) | |
| Cost | Cell counter instrument cost, maintenance, and consumables (disposable and reusable slide options) | Requires a microscope for cell counting. Reusable hemocytometers can offset recurring costs of disposable slides over time for lower throughput labs, but have a significantly higher upfront cost than disposable slides, especially for a budget conscious lab |
| Sample Volume/ Throughput | Preferable for counting multiple samples and/or replicates and for high-throughput experiments. You can also easily count just one sample at a time | Reasonable for counting a minimal number of samples; lower throughput |
| Efficiency | Automation saves time and reduces manual labor for counting, dilution, and viability calculations | Less efficient than an automated cell counter |
| Ease-of-Use | Minimal training required | More training required. Some degree of subjectivity |
| Sterility | Reduces cross-contamination risk with single-use disposable slides | Expect some level of contamination risk; user responsible for cleaning and sterilization of the hemocytometer for each sample count |
| Repeatability | Standardizes cell counting between samples, replicates, and users with a push of a button. Removes subjectivity from user to user | Relies on the user to follow lab standardization on how to count using a hemocytometer, how many quadrants to count, how to count cells on edges, etc. |
| Accuracy | Consistent and precise approach to counting | See above note on repeatability |
| Sustainability | While single-use disposable slides are less environmentally friendly, there are reusable options available for many cell counters | Environmentally friendly with reusable hemocytometer |
Visit our website to learn more about how an automated cell counter can improve cell counting in your laboratory.