ABSTRACT
Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM-H2 DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non-intuitive interpretation of laser scatter-based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub-populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, 'rolled-up' myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes 'rolling up' during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub-populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.
ABSTRACT
Airborne particulate matter (PM) has become one of the dominant pollutants with the increasing material and energy demand due to global economic growth. The main objective of this research is to provide a comprehensive receptor level characterisation of the particulate matter collected in a city environment. Particulate matter samples were collected on Tapered Element Oscillating Microbalance (TEOM) filters from five monitoring sites over a period of 1 year. An Andersen eight-stage cascade impactor was also used to collect airborne PM samples from three other locations to compare with the samples collected by TEOM. All the samples were then subjected to individual particle morphology and chemical composition analysis by SEM/EDS. Bulk chemical composition of the samples were also analysed through ICP-OES. Based on these analyses, possible sources of the PM samples were identified. The results showed that the monitoring sites in residential environments were dominated by transportation-derived particles and other migratory particulates. Monitoring sites near the city centre were dominant by particles from transportation, with biological particles abundant for the site closer to a river. The monitoring station located close to the industrial area, despite only 200 m away from a motorway, has low contribution of non-exhaust particulates from vehicles. Instead, the particulates collected from this site were dominated by industrial sources. An air dispersion modelling package was also used to model the particulate matter dispersion in the city area for the period of sampling. The results from the model showed that the points of high emissions were around industrial areas.
