Reviewer
Guillermo A. Gomez
  • Faculty, The University of Queensland
Isolation of Microglia and Analysis of Protein Expression by Flow Cytometry: Avoiding the Pitfall of Microglia Background Autofluorescence
Authors:  Jeremy C. Burns, Richard M. Ransohoff and Michaël Mingueneau, date: 07/20/2021, view: 4914, Q&A: 0

Microglia are a unique type of tissue-resident innate immune cell found within the brain, spinal cord, and retina. In the healthy nervous system, their main functions are to defend the tissue against infectious microbes, support neuronal networks through synapse remodeling, and clear extracellular debris and dying cells through phagocytosis. Many existing microglia isolation protocols require the use of enzymatic tissue digestion or magnetic bead-based isolation steps, which increase both the time and cost of these procedures and introduce variability to the experiment. Here, we report a protocol to generate single-cell suspensions from freshly harvested murine brains or spinal cords, which efficiently dissociates tissue and removes myelin debris through simple mechanical dissociation and density centrifugation and can be applied to rat and non-human primate tissues. We further describe the importance of including empty channels in downstream flow cytometry analyses of microglia single-cell suspensions to accurately assess the expression of protein targets in this highly autofluorescent cell type. This methodology ensures that observed fluorescence signals are not incorrectly attributed to the protein target of interest by appropriately taking into account the unique autofluorescence of this cell type, a phenomenon already present in young animals and that increases with aging to levels that are comparable to those observed with antibodies against highly abundant antigens.

Analysis of Random Migration of Cancer Cells in 3D
Authors:  Sai P. Visweshwaran and Alexis Gautreau, date: 01/05/2020, view: 5198, Q&A: 1
The ability of cancer cells to migrate through a complex three-dimensional (3D) environment is a hallmark event of cancer metastasis. Therefore, an in vitro migration assay to evaluate cancer cell migration in a 3D setting is valuable to examine cancer progression. Here, we describe such a simple migration assay in a 3D collagen-fibronectin gel for observing cell morphology and comparing the migration abilities of cancer cells. We describe below how to prepare the collagen-fibronectin gel castings, how to set up time-lapse recording, how to draw single-cell trajectories from movies and extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence. In our set-up, cells are sandwiched in a single plane between two collagen-fibronectin gels. This trick facilitates the analysis of cell tracks, which are for the most part 2D, at least in the beginning, but in a 3D environment. This protocol has been previously published in Visweshwaran et al. (2018) and is described here in more detail.
Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta
Authors:  Callie S. Kwartler, Ping Zhou, Shao-Qing Kuang, Xue-Yan Duan, Limin Gong and Dianna M. Milewicz, date: 12/05/2016, view: 21333, Q&A: 0
Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs from mouse aortic tissue. Conditions for maintenance and subculture of isolated SMCs and characterization of the vascular SMC phenotype are also described.
Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts
Authors:  Rodolphe Soret and Nicolas Pilon, date: 09/05/2016, view: 7478, Q&A: 0
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These “enteric” neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret et al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.
Analysis of Protein Stability by the Cycloheximide Chase Assay
Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.
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