Quantifying Metastatic Lung Colonization

10⁴ LS174T cells, either from the unsorted parental population or fractionated using the adhesion chromatography system, were suspended in 100 μL of sterile saline and each fraction was injected intravenously via the tail vein into six week old female NSG mice under isoflurane anesthesia (Jackson Labs). After 7 or 14 d, mice were sacrificed by CO₂ asphyxiation, lungs dissected, and metastatic foci counted. Afterwards, the lungs were flash frozen in optimal cutting temperature (OCT) gel in a bath of 2’methylbutane cooled in a bath of liquid nitrogen then maintained in a freezer at −80°C until cryosectioning into 10μm slices, staining with hematoxylin and eosin (H&E), and imaging with a Nanozoomer microscope (Hamamatsu Photonics). Cell densities were quanitified as previously described.^{[35 ]} Briefly, H&E stained histology images were imported into ImageJ using the NDPI tools plugin ^{[36 (link)]} and thresholded using identical parameters across all images to quantify the percent area above the threshholded background to quantify the cell density of each cryosection. All protocols for animal procedures were approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology.