TS Digest Issue | July 2024, Issue 2 | What's the Difference Between Flow Cytometry and FACS?

From the Editor| Article

The Immeasurable Impact of a Scientist

A scientist's true legacy lies in the countless researchers they inspire rather than their citation metrics.

Meenakshi Prabhune, PhD

Jul 15, 2024 | 2 min read

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A sprawling tree earns credit for producing fruits, cleansing the air, and offering a habitat for birds and small animals. But include the forest that it populated via seed dispersal, and its real influence extends beyond measure.

Scientists face a similar situation. The h-index value measures a scientist’s success based on the number of publications and citations, but that score ignores a researcher’s percolating influence. For instance, an effective group leader motivates a generation of potentially successful future scientists. On the flip side, a researcher with high accolades might create a toxic lab environment and demotivate junior researchers, deterring them from pursuing science. But those gains and losses of future discoveries are not balanced in their impact score sheet.

In a recent study, researchers found that tweeting about a paper does not increase the number of citations.¹ However, does that mean sharing information is futile? A researcher inspired to take up a new project after reading a fascinating paper or one who used a study method to troubleshoot an experiment might disagree.

When biochemist Jennifer Doudna, CRISPR pioneer at the University of California, Berkeley, spoke about the origins of CRISPR at a press conference, she credited her colleague, microbiologist Jillian Banfield, who first informed her about this bacterial immunity system and urged her to work on it. Doudna went on to characterize the CRISPR system together with Emmanuelle Charpentier, researcher at the Max Planck Institute for the Science of Pathogens, Berlin, and the duo won a Nobel Prize for their discovery in 2020. Their landmark paper has almost 20,000 citations, and CRISPR-based therapies have made their way to the clinic already.² But how should scientists quantify Banfield’s influence for sowing the seed?

Share Your Opinion

References

Branch TA, et al. PLoS One. 2024 Mar 20;19(3)
Jinek M, et al. Science. 2012;337(6096):816-821.

Infographic

A Blueprint for the Ladder of Reproduction

A trio of proteins are key building blocks for genetic exchange.

Laura Tran, PhD

Jul 15, 2024 | 1 min read

Infographic showing a stack of proteins assembling into a complex to facilitate genetic exchange of homologous chromosomes.

MODIFIED FROM © ISTOCK.COM, JANGELTUN, VORONAART, BULGAKOVA KRISTINA; DESIGNED BY ERIN LEMIEUX

Reference

Kursel LE, et al. Proc Natl Acad Sci. 2023;120(50):e2314335120.

Illustration of cells either being sorted into tubes from a cell sorter or going into a waste container from a flow cytometer.

What's in the Name| Article

What’s the Difference Between Flow Cytometry and FACS?

Flow-based methods allow researchers to collect multiparameter data from individual cells in their samples, but the fate of samples depends on the instrument.

Shelby Bradford, PhD

Jul 15, 2024 | 2 min read

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Researchers often use “flow cytometry” to refer to a method of analyzing cells on an aptly named flow cytometer.¹ However, the term also encompasses techniques that enable researchers to characterize individual components on or within a specimen, most often cells, in an entire sample rapidly and in high dimension.

In addition to cell analysis, cell sorting, sometimes referred to as fluorescence-activated cell sorting (FACS), is another common application of flow cytometry.² While these two techniques and their instruments are similar, an important distinction lies in what happens to the sample after it is analyzed.

The Specifics of Flow Cytometry

In flow cytometry, a pump draws the sample into the instrument and orients the individual cells into a single-file line for each one to pass in front of one or more lasers. Scientists instruct the computer to record information about various parameters on the cell including its size and rough composition. Detectors record information about the intensity and scattering pattern of the light after it leaves the cell. After laser interrogation, flow cell analyzers deposit all of the sample into a waste receptacle in the instrument.

FACS: To Sort, or Not to Sort

Cell sorters operate almost identically to flow cytometers up until the cell leaves the laser. After this point, cell sorters break the stream of cells into droplets by oscillating.³ The computer rapidly calculates specified qualities of the cell, and based on those parameters, applies a charge to the individual droplet holding the cell. As the now-charged droplet continues to descend away from the laser, it passes through magnetic plates that attract or repel it toward collection tubes. Sorters today can separate cells into up to six different populations.

References

McKinnon KM. Curr Protoc Immunol. 2018;120(1):5.1.1-5.1.11
Bonner WA, et al. Rev Sci Instrum. 1972;43(3):404-409
Steinkamp JA, et al. Rev Sci Instrum. 1973;44(9):1301-1310

An image of the CFX Opus Real-Time PCR System and DNA

Technology Highlight| Article

Advancing qPCR Through Innovation

Researchers obtain consistent and precise qPCR data from instruments with enhanced thermal uniformity and updated optical detection systems.

Jul 15, 2024 | 2 min read

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biorad

Scientists around the world employ quantitative real-time PCR (qPCR) to measure the amount of particular nucleic acid sequences within a sample. While this technique is rapid, sensitive, and economical, the instrument that they employ directly affects the reliability and accuracy of their data.

Within qPCR systems, reaction blocks quickly switch between heating and cooling to obtain the precise temperatures required for nucleotide denaturation, primer annealing, and polymerase-mediated extension. However, the speed at which the block changes temperature affects not only the experiment’s duration, but also the block’s thermal uniformity.¹ Furthermore, many reaction blocks lose heat faster on their edges, leading to temperature variation between sample wells and data errors.

Although stationary light sources are commonly used in qPCR instruments, they generate different light path lengths across wells in the block and consequently produce discrepancies in the data. Scientists can correct the light path differences between wells by normalizing the target-specific fluorescent signal to the signal of a passive dye, such as the ROX fluorescent dye. However, this reduces the number of sequences they can detect simultaneously using multiplexing.

Researchers require improved qPCR instruments that overcome these challenges, such as the CFX Opus Real-Time PCR System. Using an optical shuttle system to center the light-emitting diodes above each well, the instrument eliminates light path length variation and permits scientists to detect up to five targets per reaction well. This feature along with the uniform thermal performance of its innovative reaction block allows researchers to acquire accurate and reproducible measurements from all wells, while maximizing the system’s multiplexing capabilities.

Read more about this cutting-edge qPCR instrument.

What is the main problem you face when conducting qPCR experiments?

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Reference

Rogers-Broadway KR, Karteris E. Exp Ther Med. 2015;10(4):1261-1264.

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Rendering of a 3D printer working on bioprinting human brain tissue.

Article

Crafting Mindscapes of the Brain

Functional human neural tissues assembled using 3D printing technology may provide insights into neural wiring.

Laura Tran, PhD

Jul 15, 2024 | 2 min read

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Su-Chun Zhang, a stem cell biologist from Duke-National University of Singapore Medical School and the University of Wisconsin–Madison, and his team develop technology to study brain cell communication and model disease pathology.

Duke-NUS Medical School

The human brain buzzes with intricate neural highways firing signals between neurons. Understanding these networks is crucial for deciphering brain health and disease, yet existing models have had varied success. This inspired Su-Chun Zhang, a stem cell biologist at Duke-National University of Singapore Medical School and the University of Wisconsin-Madison, to design a platform to reliably model functional neural tissue.

Zhang and his team developed a bioink composed of proteins and gel polymers to 3D print brain tissues. Their findings, published in Cell Stem Cell, provide a promising tool for researchers to design and study customized brains.¹ “We developed the technology to guide human stem cells to many kinds of nerve cells, much like having all the materials to cook a dish. We just need to put them all together,” explained Zhang.

3D bioprinting enables researchers to select cell types and spatial distributions to replicate brain tissue. Traditional 3D bioprinting stacks cell layers vertically, but these structures often fail to support proper cellular connectivity.² Zhang's team crafted a Goldilocks bioink using a fibrin hydrogel that provided optimal stiffness for cell structural integrity and survival. They horizontally layered neurons and astrocytes in a pattern resembling the cerebral cortex and striatum, aiming to create functional neural circuits.

Zhang anticipated random neural connections from artificial printing, but the tissues behaved just like they do in the brain. Electrophysiological recordings and calcium imaging showed consistent neuronal communication within and across brain regions.

Massimiliano Caiazzo, a neurobiologist at Utrecht University who was not involved in the study, said, “It would be interesting to create an in vitro synaptic circuit that you can tune for disease modeling.”

“Because you can essentially control the composition of the tissue, we can artificially design models that exist and may not exist in our brain to observe how these cells talk to each other,” Zhang noted.

References

Yan Y, et al. Cell Stem Cell. 2024;31(2):260-274.e7.
Lozano R, et al. Biomaterials. 2015;67:264-273.

Infographic

Optimizing Reference Controls for Multicolor Flow Cytometry

Best practices for preventing compensation or unmixing errors.

Jul 15, 2024 | 1 min read

The quality of reference controls is critical for generating accurate compensation or unmixing matrices and correctly interpreting conventional or spectral flow cytometry data, respectively. Follow this checklist to help prevent common errors due to mismatch between reference and sample, differences in autofluorescence, inadequate gating, insufficient cell number, and use of untested and unvalidated cell surrogates.

Modified from © ISTOCK.COM, BGBLUE, RAMBO182, NADIINKO, PHOTOTECHNO; Designed by Ashleigh Campsall

Learn more about spectral flow cytometry here.

What part of flow cytometry is the most challenging?

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Reference

Spectral flow cytometry. BD Biosciences. Accessed May 8, 2024.

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A group of mussels are clustered together on a rock.

Just Curious| Article

How do Mussels Stick to Slippery Surfaces?

Biochemist Matthew Harrington investigates how bivalves produce underwater superglue.

Hannah Thomasy, PhD

Jul 15, 2024 | 2 min read

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Matthew Harrington wears a blue collared shirt and stands outside in front of a brick wall.

Matthew Harrington studies how organisms produce complex materials and how these processes could be applied to create novel materials for human use.

Laura Harrington

Intertidal mussels live life on the edge, subjected to the constant crashing of waves threatening to rip them off their precarious perches. To stay secure on the rocky shore, mussels manufacture dozens of protein-based fibers called byssal threads, which attach to the substrate via tiny discs known as byssal plaques.

This system may appear simple at first, but for McGill University biochemist Matthew Harrington, there’s more to the mussel than meets the eye. “The more you look into it, the more details you discover,” he said. “Every time I think I’m done studying this, there’s a new detail that just draws me in. It’s incredibly complex.”

While humans struggle to make effective underwater glues, mussels produce super strong, waterproof bioadhesives that cure while immersed in seawater. The strength of the adhesive, and of the byssus itself, relies in part on 3,4-dihydroxyphenylalanine (DOPA), which Harrington called “a very weird amino acid.”

DOPA forms coordination bonds with certain metal ions, like iron and vanadium.¹ This type of bond is strong, but it re-forms easily when broken, creating a material that is self-healing at the molecular level.

A blue cuboid reconstruction of mussel tissue with round plaque vesicles shown in green.

A three-dimensional reconstruction of a focused ion beam scanning electron microscopy dataset depicts adhesive secretory vesicles (green), which are an important component of the underwater glue.

Tobias Priemel

The mussel’s manufacturing process is surprisingly sophisticated. “These things are like little polymer fabrication factories,” said Harrington. His team showed that the mussel secretes DOPA-containing plaque proteins and metal storage particles containing iron or vanadium ions into microchannels in the mussel foot.² Then cilia mix the proteins and ions together within the low-pH environment of the microchannel. As the mixture is secreted, the seawater environment triggers bonding between the DOPA and the metal ions, turning the glue from a liquid to a solid.

Harrington currently explores how these mussel materials could inspire new forms of biomedical adhesives, self-healing polymers, and pH-responsive scaffolds for tissue engineering.³

What makes you curious? Submit your question for a chance to get it answered in a future "Just Curious" column.

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References

Harrington MJ, et al. Science. 2010;328(5975):216-220.
Priemel T, et al. Science. 2021;374(6564):206-211.
Rammal M, et al. ACS Appl Mater Interfaces. 2023;15(24):29004-29011.

Puzzle

Science Crossword Puzzle

Put on your thinking cap, and take on this fun challenge.

Stella Zawistowski

Jul 15, 2024 | 1 min read

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