4 – Experimental Design

A lot of microscopy assays are focused on labeling and imaging proteins. We often use antibodies against specific protein antigens or fuse a fluorescent protein to a protein of interest.  These methods cover many applications, but maybe you are interested in viral RNA, gene duplication, or need a counterstain to label the nucleus. You can’t fuse a protein to nucleic acids, so where should you start? Try these 5 assays to image nucleic acids: Nucleic Acid Dyes  Nucleic acid dyes are small molecules that increase fluorescence when bound to nucleic acids.  They can bind DNA, RNA, or both, but do…

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.

Designing microscopy experiments related to infectious diseases and antivirals can be challenging, but there's never been a more vital time than right now to design adequate microscopy experiments. The novel coronavirus (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and spread across the globe becoming the pandemic that the world is reeling with today. Currently, COVID-19  has no targeted therapies approved by the FDA, so the best coronavirus prevention happens through social distancing and good hygiene practices. However, companies are rapidly testing candidate molecules and vaccines as fast as they can. Initial tests suggest there may be some drugs that…

Experimental validation refers to the process of evaluating a panel and ensuring it’s robust. Sometimes, you're just handed a panel that you have to follow, and it might not be the best panel for your question or even your instrument. There’s a proper way to go about experimental validation, including protocols to follow. Here are 3 parts of high-quality experimental validation.

No matter how advanced or cutting-edge your study is, good science is profoundly dependent on the fundamentals. In fact, no matter your experience level, it is always good to revisit the fundamentals now and again. After all, if flow cytometry were easy, anyone could don a lab coat and get published. In reality, science is as challenging to conduct as it is exciting – that means it’s beneficial for virtually all scientists to take some time and refresh themselves on best practices. You might be surprised to realize what an impact they have on experimental quality… 

No researcher wants to discover that the results of a long, careful experiment are confounded by an uncontrolled variable. To assist in data interpretation, you must build careful controls into your experimental workflow. These controls minimize the effects of confounding variables in the experiment while helping to identify the changes related to the independent variable. When designing a flow cytometry experiment, what controls should you consider? Below are a few experimental controls that can dramatically enhance reproducibility in your flow cytometry experiments.

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

It’s not easy to improve reproducibility in your experiments. Image manipulation has become a major problem in science, whether intentional or accidental. This has exploded with the advent of digital imaging and software like Photoshop. There are even mobile applications like Instagram filters that can be used for imaging trickery. It should go without saying that image reuse/manipulation represents profound dishonesty in science – a field intended to uphold the most stringent possible standards of truthful inquiry! But what about studies with a sloppy or stunted capacity for reproduction? These, too, plague science and hinder our ability to seamlessly move…

Compensation is necessary due to the physics of fluorescence. Basically, compensation is the mathematical process of correcting spectral spillover from a fluorochrome into a secondary detector so that it is possible to identify single positive events in the context of a multidimensional panel. Good compensation requires that your controls tightly adhere to three rules. If the controls don’t meet this criteria, it will lead to faulty compensation resulting in false conclusions and poorly reproducible data. Even among flow cytometry veterans, a strong foundation is occasionally in need of a tune-up. And in a topic as complex as flow cytometry, it’s…

Reproducibility is key to the scientific method. After the results of a study are published, the community validates the findings and extends them. If the findings are not reproducible, the second step is impossible. With performable experiments increasing in complexity, and the concurrent increase in the cost of equipment and reagents to perform these experiments, it is important to find the best way to maximize the money spent on advancing research. In flow cytometry, there are many places where improvements can be made to increase the consistency and reproducibility of an experiment. The most obvious place is in the instrument,…

All flow cytometer instruments have a certain 3 components, and the way they are put together will dictate the performance of the system. As a user, you’ll be interacting heavily with these components, so you need to know both what they are and how they work. There are fluidics, optics, and electronics. The fluidics allow you to interact at the right flow rate so that your data keep a tight CV. Then you can run the same flow rate for all your samples, and you won't have different CVs for different samples. There are also different optics you can use,…

Did you know that tissues can be measured by flow cytometry? Flow cytometry is the measurement of cellular processes at the whole-cell level. This definition is useful because it includes not only flow cytometry, but any technique that measures at the level of the whole cell. Microscopy, for instance, is a great example of cytometry. But, what can be measured by flow cytometry? For one, tissues with lots of cells. When flow cytometry is practiced, the cells are broken up. Therefore, any cellular interactions within the sample are also broken up. This includes tissues, cell-to-cell contacts in tissues, and virtually…