Journal: Current protocols in immunology / edited by John E. Coligan ... [et al.]

Article Title: Isolation and Analysis of Mouse Microglial Cells

doi: 10.1002/0471142735.im1435s104

Figure Lengend Snippet: Common antibodies used for analysis of microglial cells by flow cytometry

Article Snippet: Myelin removal Buffer: Cold PBS containing 0.5% BSA (see Reagents and Solutions) Myelin Removal Beads II (Miltenyi Biotec, cat. No. 130-096-733) LS columns (Miltenyi Biotec, cat. No. 130-042-401) MS columns (Miltenyi Biotec, cat. No. 130-402-201) Sterile Transfer pipette 5mL polystyrene tubes 15 mL conical tubes MidiMACS Separator (Miltenyi Biotec, cat. No. 130-402-302) CD11b enrichment buffer (PBS, 0.5% BSA, 2mM EDTA) (see Reagents and Solutions) CD11b (Microglia) MicroBeads (Miltenyi Biotec, cat. No. 130-093-634) OctoMACS Separator (Miltenyi Biotec, cat. No.130-042-109)

Techniques:

Journal: Current protocols in immunology / edited by John E. Coligan ... [et al.]

Article Title: Isolation and Analysis of Mouse Microglial Cells

doi: 10.1002/0471142735.im1435s104

Figure Lengend Snippet: The total leukocyte population is gated from the raw data to exclude any debris that typically lies along the Y axis. It is imperative to only select the true leukocyte population in order to obtain clean plots. CD45 is sub gated from the all population into 3 different groups. CD45Hi population is made up of hematogenous derived cells that infiltrated the CNS, whereas the CD45lo population represents the CD11b+ microglial population. The CD45 negative population will be omitted from this analysis and likely contains astrocytes, or other glial and neuronal populations. Within the CD45Hi population, there are 2 populations of interest: infiltrating monocytes (CD11b+ CD11c−) and monocyte derived dendritic cells (CD11b+ CD11c+). From the CD45lo population graph CD11b by CD11c to determine the percentage of true resident microglia (CD11b+ CD11c−) and CD11b+ CD11c+ populations.

Article Snippet: Myelin removal Buffer: Cold PBS containing 0.5% BSA (see Reagents and Solutions) Myelin Removal Beads II (Miltenyi Biotec, cat. No. 130-096-733) LS columns (Miltenyi Biotec, cat. No. 130-042-401) MS columns (Miltenyi Biotec, cat. No. 130-402-201) Sterile Transfer pipette 5mL polystyrene tubes 15 mL conical tubes MidiMACS Separator (Miltenyi Biotec, cat. No. 130-402-302) CD11b enrichment buffer (PBS, 0.5% BSA, 2mM EDTA) (see Reagents and Solutions) CD11b (Microglia) MicroBeads (Miltenyi Biotec, cat. No. 130-093-634) OctoMACS Separator (Miltenyi Biotec, cat. No.130-042-109)

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Quantitative FRET Imaging to Visualize the Invasiveness of Live Breast Cancer Cells

doi: 10.1371/journal.pone.0058569

Figure Lengend Snippet: (A-B) The ECFP/FRET ratio images of a representative cell expressing the AHLR biosensor are shown at 0 min (left) and 40 min (right) after adhesion to fibronectin-coated cover glass with (B) or without (A) the pretreatment of GM6001. The same cell in panel (A) is shown in Movie S2. (C) The quantified time courses of ECFP/FRET ratio value of the MDA-MB-231 cells expressing the AHLR biosensor with or without GM6001 treatment, and that with the IV negative mutant biosensor during the adhesion process on fibronectin. (D) The averaged ECFP/FRET ratio increase of the cells in (C) 20–30 min after adhesion (MEAN±SEM). Scale bars: 10 µm. (*) indicates statistically significant difference with p-value<0.01.

Article Snippet: For the adhesion assay, fibronectin coating (from bovine plasma, Sigma, diluted to 20 µg/ml) was prepared by incubation at 37°C for 4 hours.

Techniques: Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: An in vitro correlation of metastatic capacity and dual mechanostimulation

doi: 10.1371/journal.pone.0207490

Figure Lengend Snippet: (A) Schematic of the culture well cast with soft and hard polyacrylamide hydrogels side-by-side and conjugated with bovine plasma fibronectin on the surface. A paramagnetic bead was embedded within the soft substrate and positioned approximately 0.5–1 mm away from the border of the two substrates. A multicellular spheroid was placed on the border of the two substrates. A rare-earth magnet rotated 0.5 cm below the assay plate. The rotational path of the magnet is displayed as dotted lines. (B) The assay plate prior to the placement of a spheroid onto the substrate.

Article Snippet: To facilitate cell adhesion, bovine plasma fibronectin (Sigma) at a concentration of 5μg/cm 2 was conjugated on top of the polyacrylamide substrate as described previously [ ].

Techniques:

Journal: Advanced healthcare materials

Article Title: An accessible organotypic microvessel model using iPSC-derived endothelium

doi: 10.1002/adhm.201700497

Figure Lengend Snippet: LumeNEXT organotypic approach enables microvessel modeling. (A) LumeNEXT devices consist of a culture chamber interfaced with 4 ports, across which a PDMS rod is suspended. The culture chamber is filled with a hydrogel of interest via the 2 side ports, and the rod is removed from the outlet utilizing tweezers, leaving behind a lumen in the hydrogel that can be accessed from an inlet and outlet. When viewed in cross section in culture, extracellular matrix surrounds the endothelial cell lined lumen, which has been coated for cell adhesion (i.e. fibronectin) and culture media is flowed in the luminal space. Lumens can be imaged with brightfield microscopy visualizing (B) empty fibrin lumens, as well as, lumens lined with (C) human umbilical vein endothelial cells (D) human primary kidney endothelial cells and (E) iEC-derived endothelial cells, forming model microvessels.

Article Snippet: To encourage cell adhesion, 30 µg/mL bovine fibronectin (Sigma-Aldrich, Saint Louis, Missouri) is incubated in the lumen for 20 minutes in both collagen I and fibrin devices.

Techniques: Microscopy, Derivative Assay