Counting Cells Will Save Your Flow Cytometry Experiment, If You Do It Like This

Fast, accurate or efficient – pick two. How to decide when you can’t have it all.

The hemocytometer is considered the gold standard for cell counting. Invented by Louis Charles Malassez, this precision etched microscope slide can allow the researcher to count their cells under the microscope with amazing accuracy. It is inexpensive, relative to other methods, but is by no means the most efficient or fast method out there.

Counting 1

The single biggest key to using a hemocytometer is training, training and more training. Since the investigator is visually inspecting the cells within a boundary, the rules of what cells to count and what to exclude on those boundaries becomes critical.

If counting more than one sample, proper cleaning of the hemocytometer is a second critical step.

If the cleaning solution is not removed completely, it can cause cell lysis and thus lower than expected cell numbers.

The bias that an investigator brings to the hemocytometer and the slow speed for counting cells is why many users are moving toward automated counting methods. These can be divided into three major categories – image based, impedance based and cytometry based.

Image-based methods (such as the implemented in the Cellometer, the T20, and the Countess) all involve a system that takes a picture of a defined area (using a proprietary slide) and identifies the cells based on the relative size. These systems also can count ‘dead’ cells using Trypan Blue (like can be done with the hemocytometer). All of these systems are relatively rapid at counting the cells, and accurate within certain size ranges.

Counting 2

Wallace Coulter discovered and patented the impedance principle for measuring cells in solution. This technique is still used in the clinical setting in cell counters today. It is also a very accurate way to measure the number and size of cells. Impedance measurements, as commercialized in the Coulter counter, the Scepter and the Casy counter, are also very accurate, require a diluted sample (especially the Scepter). Dead cells are measured based on their size (smaller than normal cells). The Scepter has the advantage of being a hand-held, pipet like device, making it amenable to rapid cell counting in a tissue hood.

Counting 3

The use of the flow cytometer as a cell counter requires either a pump driven system, which allows for a very accurate measurement of the volume of sample. It is a simple calculation to determine the concentration of the sample.

Instruments like the Accuri and the Guava are excellent tools for counting cells. Additionally, one can use a cell impermeant dye, like PI or 7AAD, for measuring the dead cells. This is more accurate than using Trypan Blue and visually inspecting the ‘blueness’ of the cells.

Counting 4

With a displacement (pressure system) like most commonly available instruments, an extra step is required. It is not possible to have a very accurate volume measurement, so a counting particle must be added to the sample. This requires a very accurate pipet to dispense the counting particle into the sample. Once the sample is run on the flow cytometer, the number of counting particles can be measured and ratio of collected particles to total particles can be used to determine the original count in the sample. This method is very good for high throughput applications, typically integrated into the sample at the end – rather than a simple counting method at the beginning of an assay.

Regardless of how you count your cells, make sure that it is done consistently and reproducibly.

In summary, counting cells is essential to flow cytometry because:

1. Know the percentage of your target cells to determine how many cells you need to start with.

2. Perform a preliminary experiment to determine how many cells are lost in the process.

3. Based on #1 and the losses of #2, that will determine the minimum number of cells that must be stained

4. Each of the four different methods for counting cells has its strengths and weaknesses. Remember the old adage fast, accurate or efficient – pick two and that dictates the third.

Houston, U.S. (Register Online)

Aarhus, Denmark (Register Online)

Louisville, U.S. (Register Online)

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Similar Articles

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

By: Tim Bushnell, PhD

In the flow cytometry community, SPADE (Spanning-tree Progression Analysis of Density-normalized Events) is a favored algorithm for dealing with highly multidimensional or otherwise complex datasets. Like tSNE, SPADE extracts information across events in your data unsupervised and presents the result in a unique visual format. Given the growing popularity of this kind of algorithm for dealing with complex datasets, we decided to test the SPADE algorithm in 5 software packages, including Cytobank, FCS Express, FlowJo, R, and the original, free software made available by the author of SPADE. Which was the fastest?

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

By: Tim Bushnell, PhD

FlowJo is a powerful tool for performing and analyzing flow cytometry experiments, if you know how to use it to the fullest. This includes understanding embedding and using keywords, the FlowJo compensation wizard, spillover spreading matrix, FlowJo and R, and creating tables in FlowJo. Extending your use of FJ using these hacks will help organize your data, improve analysis and make your exported data easier to understand and explain to others. Take a few moments and explore all you can do with FJ beyond just gating populations.

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

By: Tim Bushnell, PhD

It is necessary to sort through hundreds of thousands or millions of cells to find the few events of interest. With such low event numbers, we move away from the comfortable domain of the Gaussian distribution and move into the realm of Poisson statistics. There are 3 points to consider to build confidence in the data that the events being counted are truly events of interest and not random events that just happen to fall into the gates of interest.

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

By: Tim Bushnell, PhD

Preparing for rare event analysis requires an understanding of the power and limitation of the instrument to be used. From how fast to run the fluidics, to how the signal is processed to the number of gates that can be used in the sorting experiment, each factor impacts the outcome of the experiment.

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

With the added emphasis on reproducibility, it is critical to look at every step where experiments can be improved. No single step makes an experiment more reproducible, rather it is a process, making changes at each stage that leads to reproducibility. Antibodies comprise a critical component that needs to be reviewed. As Bradbury et al. in a commentary in Nature pointed out, the global spending on antibodies is about $1.6 billion a year, and it is estimated about half of that money is spent on “bad” antibodies. This does not include the additional costs of wasted time and effort by…

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

By: Tim Bushnell, PhD

Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease. Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity. Calcium experiments can be very informative, and with the advent of cheaper UV lasers, more and more researchers can use ratiometric measurements to evaluate the signaling processes in phenotypically defined populations.

How to Perform Doublet Discrimination In Flow Cytometry

How to Perform Doublet Discrimination In Flow Cytometry

By: Tim Bushnell, PhD

You are probably familiar with the term, “doublet discrimination” or “doublet exclusion”, and have likely included this flow cytometry measurement into at least some (if not all) of your gating strategies. Even though you may utilize this important gating strategy, you may not have had the chance to delve deeper to explore exactly what doublets are and why it’s critical to exclude them. This article aims to give you insight on the what, why, and how of doublet discrimination.

4 Considerations For Assessing Protein Phosphorylation Using Flow Cytometry

4 Considerations For Assessing Protein Phosphorylation Using Flow Cytometry

By: Tim Bushnell, PhD

For those working in the signaling field, having the ability to take a sample and phenotypically identify it, while knowing what is happening inside the cell to the target molecules of choice opens up a host of new opportunities. These assays are amenable to high throughput setup, meaning that biologically relevant outcomes in pre-clinical drug discovery can be measured directly. All told, with a little forethought, some careful planning and validation, and our helpful tips, phosphoflow assays are within your reach.

5 Essential Calculations For Accurate Flow Cytometry Results

5 Essential Calculations For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

Flow cytometry is a numbers game. There are percentages of a population, fluorescence intensity measurements, sample averages, data normalization, and more. Many of these common calculations are useful, but surrounded by misconceptions. This primer will help you decide which calculation to use, when to use it, and how to interpret the results.

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.