The THP-1 monocyte cells were seeded on a fibronectin-functionalized glass slide (Corning #2980–246) at 80%−90% confluence contained by a rubber gasket (Grace Bio-Labs #JTR8R-2.5). To differentiate the THP-1 monocytes into macrophages, the cells were incubated with 10ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich #P8139) in RPMI with BME for 24 hours. The media was then replaced with fresh RPMI with BME and incubated for an additional 24 hours. Differentiation was confirmed visually based on changes in adherence and morphology.
The macrophages were dosed with 1ug/mL LPS (Sigma Aldrich #L4524) in RPMI with BME for 3 hours. Cells were rinsed with warm 1X PBS, fixed with fresh 4% formaldehyde (Thermo #28908) in 1X PBS for 10 minutes at room temperature, and then permeabilized with 70% ethanol overnight at −20°C. The primary probe library (Spatial Genomics) was added to the sample in a flow chamber provided by Spatial Genomics and incubated overnight at 37°C. The sample was washed several times with primary wash buffer (Spatial Genomics). The nuclei of the sample were stained with staining solution (Spatial Genomics).
The macrophage sample was imaged with the Spatial Genomics Gene Positioning System (GenePS). Image tiling and secondary probe staining were performed programmatically by this instrument.
The macrophages were dosed with 1ug/mL LPS (Sigma Aldrich #L4524) in RPMI with BME for 3 hours. Cells were rinsed with warm 1X PBS, fixed with fresh 4% formaldehyde (Thermo #28908) in 1X PBS for 10 minutes at room temperature, and then permeabilized with 70% ethanol overnight at −20°C. The primary probe library (Spatial Genomics) was added to the sample in a flow chamber provided by Spatial Genomics and incubated overnight at 37°C. The sample was washed several times with primary wash buffer (Spatial Genomics). The nuclei of the sample were stained with staining solution (Spatial Genomics).
The macrophage sample was imaged with the Spatial Genomics Gene Positioning System (GenePS). Image tiling and secondary probe staining were performed programmatically by this instrument.
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