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.
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.
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.
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.
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.
ABOUT 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.More Written by Tim Bushnell, PhD