In Vitro Cellular Cluster Generation

In vitro clusters were generated as previous described^{17 (link)}. Briefly, adherent cells (PD7591 and PD798) were trypsinized and cultured in a 6 cm suspension culture dish with rocking for 12–16 h in a 37 °C incubator at 5% CO₂ at a density of approximately 1 million cells per ml in 5 ml media. 100 μl of cell suspension was then spiked into 0.5 ml of mouse blood and subjected to the sample preparation workflow described above. 1000 cells (events) were sorted into 4 wells of a 12-well plate for each sample. 5–6 individual images were collected per well and the counts were added to obtain a representative count for each well. The mean for 4 wells was reported. In order to assess the distribution of clusters prior to sorting, 1 ml of the cell suspension prior to spiking into blood was imaged in a 6 cm dish. 7 representative images were collected and size and number of clusters were quantified. All wells were stained with Hoechst 33342 and imaged on an Olympus IX71 inverted multicolor fluorescent microscope.