Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
If you work with eukaryotic cells in culture, it’s a sure bet you’ll need to count them, at least occasionally. You need to know how many cells you have for passaging and expansion, for transfection, and, really, for everything else as well.
Cell counters can be either manual or automated. The manual (and least expensive) approach is trypan blue staining using a dedicated slide called a hemocytometer and a microscope. Trypan blue stains dead cells, so by counting the unstained cells in a given volume, researchers can compute the live cell concentration in the original solution. But this method is labor-intensive, tedious, and prone to mistakes – hence the automated alternatives.
Automated instruments are available in three basic designs, “Coulter counters,” image-based counters, and flow cytometers. Accessory reagents are also available. Read on to find out what you need to know before making a purchasing decision.
Coulter counters
So-called “Coulter counters” are based on the “Coulter Principle” (also called the “electrical sensing zone” method). Such instruments have a probe that is inserted into a solution containing the particles to be counted. This probe contains two electrodes separated by a small hole through which the fluid can pass. As particles pass through the aperture, they disrupt the impedance between the two electrodes. The instrument records those events, and usually particle size data as well, so that researchers can determine, for instance, how many cells of a particular type are present.
Beckman Coulter sells several instruments based on the Coulter Principle, including both the new Z2™ and Multisizer 4™ Coulter Counter® instruments. Also based on the Coulter Principle are the CASY Cell Counter and Analyzer Model TTC from Roche Applied Science, the microfluidics-based Moxi Z™ from ORFLO, and the Scepter™ cell counter from EMD Millipore.
Most Coulter Principle-based instruments are desktop units. But Millipore’s Scepter 2.0 cell counter is actually a handheld pipettor that quantifies cells as they are drawn up into a specialized consumable pipette tip probe, making the system useful in space-constrained environments such as culture hoods. (The Scepter can count cells measuring from 4-to-25-microns in diameter, but other counters cover different size ranges. For instance, the Multisizer 4 handles particles from 0.4 to 1,600 microns.)
Imaging-based counters
Instruments in this category count cells via image analysis, and can be either slide-based like a hemocytometer, or flow-based. Some such systems allow users to actually view the cells being counted (a kind of quality control measure), and can even store those data if desired.
Bio-Rad Laboratories’ slide-based TC20™ Automated Cell Counter, Logos Biosystems’ Luna™ Automated Cell Counter. Life Technologies’ Countess™ Automated Cell Counter are all slide-based counters that use also counts via trypan blue staining to quantify both live and dead cells. on a slide, for cells up to about 60-microns in size.
ChemoMetec's NucleoCounter® Automated Cell Counting System uses a disposable plastic microfluidic consumable, called a NucleoCassette™, which is preloaded with a fluorescent nuclear stain, propidium iodide (PI). PI stains nuclei of dead cells. “Because the NucleoCounter detects signals from the stained nuclei, and not the cell, the system operates without regard to cell size or morphology,” according to product literature. “It is therefore capable of easily counting a variety of mammalian culture types including CHO, hybridomas and adipocytes, as well as yeast cultures, without requiring calibration.”
Nexcelom Bioscience’s Cellometer® systems are also slide-based, and can run both brightfield and two-color fluorescence assays (depending on the model). For instance, the Cellometer Mini is a trypan blue-based counter, whereas the Cellometer Vision can use fluorescent nuclear dyes such as propidium iodide and acridine orangeto differentially stain both live and dead cell nuclei.
Roche Applied Sciences’ high-throughput Cedex HiRes Analyzer and Beckman Coulter’s Vi-CELL™ analyzers are flow-based imaging counters. The Cedex HiRes can stain and count up to 20 samples at a time by trypan blue staining, and is intended mostly for industrial applications. The Vi-CELL XR features an autosampler to handle up to 12 samples, and can handle cells as small as 2-microns in diameter, making it useful for non-mammalian applications (such as counting yeast).
Flow-based systems
Flow cytometers are not normally capable of acting as absolute cell counters, as they count events, but not the volume those events were counted in. Thus, they typically report relative values – that is, that 25% of the cells in a given sample were, say, B cells.
To make a flow cytometer behave as a true cell counter, you need to spike your sample with control fluorescent counting beads, such as Life Technologies’ CountBright™ Absolute Counting Beads, DakoCytomation’s CytoCount™ beads, or Thermo Scientific’s Cyto-Cal particles.
Some flow cytometers can provide direct counts, though, thanks to fluidic systems that move defined volumes of liquid. These include EMD Millipore’s guava easyCyte™, BD Biosciences’ Accuri™ C6, Life Technologies’ Attune® Acoustic Focusing Cytometer, and EMD Millipore’s Muse™ Cell Analyzer.
If you’re feeling overwhelmed, don’t. You have are many choices, but all of them are easier and faster than traditional hemocytometry. Long story short: Go ahead and focus on your science; you never need worry about counting again.
The image at the top of the page is from EMD Millipore.