Review: An introduction to automated flow cytometry gating tools and their implementation

An introduction to automated flow cytometry gating tools and their implementation

Chris Verschoor, Alinia Lelic, Jonathan Bramson & Dawn ME Bowdish

Frontiers in Immunology
Front. Immunol., 27 July 2015 | http://dx.doi.org/10.3389/fimmu.2015.00380

Current flow cytometry (FCM) reagents and instrumentation allow for the measurement of an unprecedented number of parameters for any given cell within a homogenous or heterogeneous population. While this provides a great deal of power for hypothesis testing, it also generates a vast amount of data, which is typically analyzed manually through a processing called “gating.” For large experiments, such as high-content screens, in which many parameters are measured, the time required for manual analysis as well as the technical variability inherent to manual gating can increase dramatically, even becoming prohibitive depending on the clinical or research goal. In the following article, we aim to provide the reader an overview of automated FCM analysis as well as an example of the implementation of FLOw Clustering without K, a tool that we consider accessible to researchers of all levels of computational expertise. In most cases, computational assistance methods are more reproducible and much faster than manual gating, and for some, also allow for the discovery of cellular populations that might not be expected or evident to the researcher. We urge any researcher who is planning or has previously performed large FCM experiments to consider implementing computational assistance into their analysis pipeline.

For access to the .pdf click here.

 

Manuscript: The evolution of the scavenger receptor cysteine-rich domain of the class A scavenger receptors

Do you work out? Cause you’re built like a rock! A rock like Dwayne “The Rock” Johnson! You have an impenetrable body thanks to your complex immune system. So how did you get such a sophisticated immune system?

In the Bowdish lab, we do more than just macrophage biology; we also study the evolution of the immune system! The scavenger receptors are a group of receptors that play an important role in your immune system by binding harmful bacteria. Our most recent publication by Yap et al., looks at how these receptors evolved and how evolution has changed their function. These receptors are found in various forms of life such as sharks, frogs, and mammals, but the function and appearance of these receptors has changed over time. Check out the open access….

Stearns JC, Davidson CJ, McKeon S, Whelan FJ, Fontes ME, Schryvers AB, Bowdish DM, Kellner JD, Surette MG. Culture and molecular-based profiles show shifts in bacterial communities of the upper respiratory tract that occur with age. ISME J. 2015 May;9(5):1268. doi: 10.1038/ismej.2015.49.

Stearns JC, Davidson CJ, McKeon S, Whelan FJ, Fontes ME, Schryvers AB, Bowdish DM, Kellner JD, Surette MG. Culture and molecular-based profiles show shifts in bacterial communities of the upper respiratory tract that occur with age. ISME J. 2015 May;9(5):1268. doi: 10.1038/ismej.2015.49.

Abstract: The upper respiratory tract (URT) is a crucial site for host defense, as it is home to bacterial communities that both modulate host immune defense and serve as a reservoir of potential pathogens. Young children are at high risk of respiratory illness, yet the composition of their URT microbiota is not well understood. Microbial profiling of the respiratory tract has traditionally focused on culturing common respiratory pathogens, whereas recent culture-independent microbiome profiling can only report the relative abundance of bacterial populations. In the current study, we used both molecular profiling of the bacterial 16S rRNA gene and laboratory culture to examine the bacterial diversity from the oropharynx and nasopharynx of 51 healthy children with a median age of 1.1 years (range 1–4.5 years) along with 19 accompanying parents. The resulting profiles suggest that in young children the nasopharyngeal microbiota, much like the gastrointestinal tract microbiome, changes from an immature state, where it is colonized by a few dominant taxa, to a more diverse state as it matures to resemble the adult microbiota. Importantly, this difference in bacterial diversity between adults and children accompanies a change in bacterial load of three orders of magnitude. This indicates that the bacterial communities in the nasopharynx of young children have a fundamentally different structure from those in adults and suggests that maturation of this community occurs sometime during the first few years of life, a period that includes ages at which children are at the highest risk for respiratory disease.

Yap N, Whelan FJ, Bowdish DM and Golding B (2015). The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors. Front. Immunol. 6:342. doi: 10.3389/fimmu.2015.00342

Yap N, Whelan FJ, Bowdish DM and Golding B (2015). The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors. Front. Immunol. 6:342. doi: 10.3389/fimmu.2015.00342

Abstract

The class A Scavenger Receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria such as Streptococcus pneumoniae, and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a Scavenger Receptor Cysteine Rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the Macrophage Associated Receptor with COllagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO’s SRCR domain which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together these findings indicate a common origin of the SRCR domain within the cA-SRs; however different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

Love RJ, Patenaude M, Dorrington M, Bowdish DM, Hoare T, Jones KS. An investigation of scavenger receptor A mediated leukocyte binding to polyanionic and uncharged polymer hydrogels. J Biomed Mater Res A. 2015 May;103(5):1605-12. doi: 10.1002/jbm.a.35297.

Love RJ, Patenaude M, Dorrington M, Bowdish DM, Hoare T, Jones KS. An investigation of scavenger receptor A mediated leukocyte binding to polyanionic and uncharged polymer hydrogels. J Biomed Mater Res A. 2015 May;103(5):1605-12. doi: 10.1002/jbm.a.35297.

Abstract
Cell adhesion to biomaterials can be mediated in part by mechanisms aside from the traditionally recognized opsinization and integrin binding mechanisms. In this study, we investigated the role of scavenger receptor A (SR-A) in leukocyte binding to a series of well-controlled polyanionic and uncharged hydrogels based on a poly(N-isopropylacrylamide) backbone. The hydrogels were injected in the peritoneal cavity of SR-A knockout (KO) and wild-type mice using a minimally invasive procedure and allowed to set in situ. After 24 h, the hydrogels were recovered and analyzed, the peritoneal cavity was lavaged, and cytokine concentrations were assessed by ELISA. The polyanionic hydrogels retrieved from the KO animals were found to be completely devoid of adherent leukocytes, which were present in other materials regardless of the mouse strain in which they were injected. Results from a subsequent in vitro cellular adhesion study with a RAW264.7 cell line failed to yield a similarly definitive role for SR-A in the cellular binding of a polyanionic hydrogel. Taken together, the results of this study show that SR-A mediates leukocyte adhesion to a polyanionic hydrogel in the peritoneal cavity, but other adhesion mechanisms contribute to cellular binding in vitro. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1605–1612, 2015.

The Bowdish lab has a new PhD student! Congrats Avee!

 

 

Avee Naidoo successfully passed her transfer exam today. She now joins the ranks of Fan Fei, Mike Dorrington, Kyle Novakowski, Dessi Loukov, Sara Makaremi, and Pat Schenck as part of the Bowdish lab PhD posse. Well done Avee!

 

Avee-med

Becoming a PhD student in the Bowdish lab is a family affair. Junior lab members  sent their favourite stuffie in to wish Avee luck. It must have worked as she aced it!Becoming a PhD student in the Bowdish lab is a family affair. Junior lab members sent their favourite stuffie in to wish Avee luck. It must have worked as she aced it!

...and a little lab humour. We have both skilled scientists (see picture at bottom) and skilled artists (see whiteboard) in our lab.

…and a little lab humour. We have both skilled scientists (see picture at bottom) and skilled artists (see whiteboard) in our lab.

The Bowdish lab attends the 14th Annual Buffalo Immunology Conference.

This year our lab is taking the Buffalo Immunology Conference by storm!

Nick Yap (MSc candidate with Dr. Brian Golding) was chosen to give a talk “The evolution of the scavenger receptor cysteine-rich domain of the class A scavenger receptor family”. Dessi Loukov (PhD candidate) will be speaking on “MacrophAGING: The effects of chronic inflammation on macrophage anti-microbial immunity” . Dawn will be giving a talk on “The aging microbiome drives age‐associated
inflammation” while many other members of the team give poster presentations.