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So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

Developing variant calling and analysis pipelines for NGS sequenced data have become a norm in clinical labs. These pipelines include a strategic integration of several tools and techniques to identify molecular and structural variants. That eventually helps in the apt variant annotation and interpretation. This blog will delve into the concepts and intricacies of developing a “variant calling” pipeline using GATK. “Variant calling” can also be performed using tools other than GATK, such as FREEBAYES and SAMTOOLS.  In this blog, I will walk you through variant calling methods on Illumina germline RNASeq data. In the steps, wherever required, I will…

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

Clinical trials are studies designed to test the novel methods of diagnosing and treating health conditions – by observing the outcomes of human subjects under experimental conditions.  These are interventional studies that are performed under stringent clinical laboratory settings. Contrariwise, non-interventional studies are performed outside the clinical trial settings that provide researchers an opportunity to monitor the effect of drugs in real-life situations. Non-interventional trials are also termed observational studies as they include post-marketing surveillance studies (PMS) and post-authorization safety studies (PASS). Clinical trials are preferred for testing newly developed drugs since interventional studies are conducted in a highly monitored…

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

In the first blog of this series, we explored the power of sequencing the genome at various levels. We also dealt with how the characterization of the RNA expression levels helps us to understand the changes at the genome level. These changes impact the downstream expression of the target genes. In this blog, we will explore how NGS sequencing can help us comprehend DNA modification that affect the expression pattern of the given genes (epigenetic profiling) as well as characterizing the DNA-protein interactions that allow for the identification of genes that may be regulated by a given protein.  DNA Methylation Profiling…

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

Why is Next Generation Sequencing so powerful to explore and answer both clinical and research questions. With the ability to sequence whole genomes, identifying novel changes between individuals, to exploring what RNA sequences are being expressed, or to examine DNA modifications and protein-DNA interactions occurring that can help researchers better understand the complex regulation of transcription. This, in turn, allows them to characterize changes during different disease states, which can suggest a way to treat said disease.  Over the next two blogs, I will highlight these different methods along with illustrating how these can help clinical diagnostics as well as…