5 – Flow Cytometry

While we normally cover purely technical content for application in your lab, this week’s article is a bit different. I want to talk about a highly effective career option that I know from many years of direct experience: Shared Resource Laboratories (SRLs). Also known as “core facilities,” these research hubs represent a significant investment—by some number of institutions—in personnel and resources. In an SRL, staff and directors possess advanced training, allowing them to support resident researchers.

Histological stains that have an affinity for specific cellular components have been in use since at least the 1770s when John Hill used carmine to study tissues. Stain variety exploded during the 1800s with German dye manufacturers, such as BASF, developing aniline, methylene blue, and eosin. Eosin is still in use today with hematoxylin for H&E staining.  Since the advent of immunofluorescence and fluorescent protein tagging, which provides very specific labeling, dyes have been relegated to only the most basic imaging. If you don’t need specific proteins labeled, dyes can be a cheap and useful alternative offering simple sample preparation…

The global bacterial biomass has been estimated to be 5x1030, which is significantly higher than plants and animals. We are intimately dependent on bacteria for processing waste, producing vitamin B12, fixing nitrogen and so much more.  While some bacteria are known pathogens, most are not.  These organisms live in all environments from the soil to hot springs to deep thermal vents.  

Analog instruments processed data differently than the current generation of digital instruments. With analog systems, if the populations were “off-scale,” especially at the low end of the scale, the data accumulated in the first channel. When setting voltage, highly autofluorescent cells would drive the voltage, and it was not uncommon for less autofluorescent cells on the axis to get compressed. Unfortunately, due to the way the data were plotted, this side effect was not always clear to observers.

What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”

Because mass cytometry allows users to characterize masses so effectively, data can be normalized much more efficiently than what traditional fluorescent flow will permit. If there is no working CyTof at your institution, you can still partner with CyTof-friendly research institutions that have the technology on hand. And because the samples are fixed, you can ship them overnight. This way, they will be analyzed for you. Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should…

A lot of the troubleshooting is focused on fluidics issues. If you sit down and think about your workflow, and how you might want to add a couple of little tweaks here and there which will ultimately help you improve the quality of your data as well as aid you in identifying issues before they become problems your troubleshooting will be much smoother. Consider these three things, what do you before you start collecting data, ensure you have appropriate plots of time vs fluorescence for each of the lasers your using and apply appropriate gating procedures.

For those new to flow cytometry, compensation is confusing at best and terrifying at worst. Likewise, those who have been doing flow cytometry since the analog ages may be holding on to practices that, while suited to the analog instruments, should be left to the annals of history. As such, a lot of time is spent discussing compensation and the best practices for this critical process. There are 3 rules that guide proper compensation, and they’ve been written about extensively since they first appeared in the “Daily Dongle” in 2011. Here, we will review the classic rules and expand upon…

Congratulations, your grant has been funded! Next comes generating data and publishing papers. What was that hypothesis again? It must be in the grant somewhere, right? To avoid even the appearance of HARKing — Hypothesizing After The Results Are Known — it is important to start the statistical analysis process even before the first experiments are performed. This process consists of 5 steps: setting the null hypothesis, establishing a threshold, performing the experiments, performing the statistical tests, and communicating the results. Walk with us as we discuss these steps in an example workflow.

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.

There is a lot of preparatory work that must be done before the first flow cytometry experiment can be attempted. Each step builds upon the previous one and extends where the assay is going. Be prepared for some trial and error in this process, and don’t expect perfect results the first time around. An educated user is a good user, and makes the SRL staff’s job that much easier. The partnership between investigator and SRL staff is a rewarding one, when both parties work together to achieve the ultimate goal of generating excellent data and sort results that help answer…

Here, we cover 5 lessons from the trenches of flow cytometry looking at important aspects of how best practices have changed over time, which practices need to be adopted, and which are outdated. Put those old, coffee-stained protocols away and take advantage of the best practices for digital instruments to write new and improved ones (coffee stains optional). Your data will thank you.