Dmi 6000 microscope

Ex vivo isolated macrophages or starved RAW246.7 cells (40000 cells/well) were placed into the upper chamber of a 24-multiwell insert system with 5-μm pore size polycarbonate filter (Corning). Cell migration was stimulated with VEGF-A or PLGF-1 (100 ng/mL) added to the starvation medium into the lower chamber, in presence of iVR1 (2 μM, 10 μM or 50 μM) or CP (50 μM). After 24 h for RAW246.7, or 6 h for peritoneal macrophage, cells on the top of the filter were removed and those on the bottom side were stained with DAPI. Images were recorded on Leica DMI 6000 microscope equipped with Hamamatsu Orca R2 camera. Single cells were counted using Tile Scan Macro of LAS AF Software (Leica).

Microscopy was completed using a Leica DMI 6000 microscope equipped with a Hamamatsu Orca AG camera, a Sutter DG4 light source, a Ludl emission filters wheel with Chroma bandpass emission filters (Leica Microsystems GmbH). For each frame, Z-stacked images (0.2 μm across 7 μm) were obtained using Volocity 4.3.2 (PerkinElmer) using a 63× objective without binning and 3 s exposure for GFP fluorescence. Images were analyzed using NIH FIJI ImageJ software (https://imagej.nih.gov/ij/download.html) (60 ).
Microscopy experiments were completed in triplicate, and each replicate consisted of at least 100 cells. The number of total cells and number of cells that form SGs were determined by manual counting of stacked images. From these values, the percentage of cells that form SGs was determined. Statistics was completed using an online t test calculator by GraphPad Prism (https://www.graphpad.com/quickcalcs/ttest1.cfm). ANOVA was utilized, with a p-value of <0.05 representing statistical significance.