Stem cells, circulating tumor cells, and minimal residual disease in cancer patients were all discovered through the power of rare event flow cytometry. When preparing for rare event analysis, sample preparation and data analysis must be taken into account at the beginning. How will we stain our cells? How will we analyze our cells? What controls will we use to help us identify our rare events? What statistical methods do we use to analyze our results? Here are 5 procedural limitations that impact the quality of rare event flow cytometry data and how to optimize your assay to get the…
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.
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.
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…
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.
Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer. Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control,to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.
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.
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.
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.
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.