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When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design. When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell,…

In an ideal world, we would be able to use fluorophores that don’t have any overlap in emission spectra and autofluorescence wouldn’t obscure your signal. Unfortunately, we don’t live in such a world and often have to use two closely related dyes – or contend with fluorescent molecules that are innately part of our sample. Fluorescent molecules include chlorophyll, collagen, NADPH, and vitamin A.  One example that I recently encountered was developing a new probe for lipids. The reviewers requested a direct comparison of the new dye to Nile Red in the same sample. Both dyes would localize to the…

Next Generation Sequencing (NGS) is a rapidly evolving and widely used method worldwide in both academic and non-academic settings. One of the most valuable aspects of NGS is producing millions of sequenced reads with diverse read lengths from small amounts of input DNA. NGS methods are extremely versatile; producing reads as short as 75 bp, as seen in SOLiD sequencing, to long reads ranging upwards of 1000bp in the case of Pyrosequencing.  Both long and short reads fill a unique niche for researchers. Longer reads generated from NGS are excellent for genomic rearrangement and genome assembly projects; especially when there…

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 not differentiate specific sequences.

The process of fluorescence starts with a photon of light, which causes the excitation of a compound that releases the absorbed energy by emitting a photon of light. This process is very rapid (typically occurring in nanoseconds), and the emitted photon is of higher wavelength (thus lower energy) than the exciting photon. Here is your guide to how full spectrum cytometry works.

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 with no or basic microscope techniques. Today we will discuss 7 dyes you should know for use in a laboratory setting.

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.

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 could be repurposed for COVID-19 treatment. Repurposed drugs have already shown their safety, so clinical trials need to be conducted for effectiveness only. 

One question that I get asked on a regular basis is what flow cytometer should I purchase?  It’s not as simple as you might imagine. In fact, you need to treat this process as carefully as you would a valuable experiment. There are a lot of variables, and if you’re not asking yourself a huge list of questions, you may miss something critical that will result in the instrument being less than desired. This is a shortlist of questions to ask as you go about the process – your 3-part pocket guide to acquiring the flow cytometer that’s right for you. 

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.