Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the degradation of the extracellular matrix and the isolation of single cells, allowing the generation of high‐quality flow cytometry data. Cells and the extracellular matrix contain various proteins and other structures which must be considered when designing a tissue digestion protocol to preserve the viability of cells and the presence of relevant antigens while digesting matrix components and cleaving cell–cell junctions. Evaluation of the single cell suspension is essential before proceeding with the labeling of the cells as high viability and absence of cell debris and aggregates are critical for flow cytometry. The information presented should be used as a general guide of steps to consider when preparing a single cell suspension from solid tissues for flow cytometry experiments. © 2018 International Society for Advancement of Cytometry

Angiogenesis extends pre-existing blood vessels to improve oxygen and nutrient delivery to inflamed or otherwise hypoxic tissues. Mitochondria are integral in this process, controlling cellular metabolism to regulate the proliferation, migration, and survival of endothelial cells which comprise the inner lining of blood vessels. Mitochondrial Complex III senses hypoxic conditions and generates mitochondrial reactive oxygen species (mROS) which stabilize hypoxia-inducible factor (HIF-1α) protein. HIF-1α induces the transcription of the vegfa gene, allowing the translation of vascular endothelial growth factor (VEGF) protein, which interacts with mature and precursor endothelial cells, mobilizing them to form new blood vessels. This cascade can be inhibited at specific points by means of gene knockdown, enzyme treatment, and introduction of naturally occurring small molecules, providing insight into the relationship between mitochondria and angiogenesis. This review focuses on current knowledge of the overall role of mitochondria in controlling angiogenesis and outlines known inhibitors that have been used to elucidate this pathway which may be useful in future research to control angiogenesis in vivo.

Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here, a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45 αSMA , indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts. © 2018 International Society for Advancement of Cytometry.

The occurrence of new blood vessel formation in the airway wall of asthma patients was reported more than a century ago. It was long thought that angiogenesis in asthma was an epiphenomenon of airway inflammation. Therefore, little research has been performed on the role of endothelial cells in this disease. We are moving away from this misconception as an increasing number of clinical studies and findings in murine models of asthma demonstrate a causal link between angiogenesis in the airway and genesis of allergic asthma. In this chapter, we review the evidence supporting key roles for the endothelium and other angiogenic cells in the pathogenesis of asthma.

Introduction: Injury to the posterolateral corner (PLC) of the knee often requires surgical reconstruction. There remains no consensus on treatment for PLC injury, and, therefore, it is imperative to have a reproducible injury model to improve the general knowledge of PLC injuries. A novel cadaveric model of isolated PLC injury is proposed and evaluated using radiographic parameters as well as gross dissection. Material and methods: All protocols were reviewed by the Human Investigation and Research Committee of the home institution and were approved. Translational force in a defined posterior and lateral direction was applied to cadaveric native knees to induce PLC injury. Varus and recurvatum stress fluoroscopic imaging was obtained of each specimen before and after the injury model. Lateral joint distance and recurvatum angle after stress was measured on each image via picture archiving and communication software (PACS) imaging software. After the injury model, injured structures were assessed via saline loading and gross dissection. Any specimens found to be fractured were excluded from the analysis of stress radiography. Results: A total of 12 knees underwent testing and 6/12 successfully induced PLC injury without fracture. The lateral capsule was torn in every specimen. The popliteofibular ligament (PFL) was torn in 83% of specimens and the fibular collateral ligament (FCL) in 66.7% of specimens. The median lateral gapping after injury under varus stress radiography was 5.39 mm and the median recurvatum angle after injury was 14.25°. Radiographic parameters had a direct relationship with a number of structures injured. Conclusions: This is the first successful cadaver model of PLC injury. The lateral capsule was injured in every specimen emphasizing the importance of this structure to the PLC.