2.3. MCF7 cell capture and spike‐in experiment

MCF7 cells were incubated for 10 min with 2 mm calcein AM (Life Technologies) in cell culture media. The cells were centrifuged and washed once in PBS, counted with a hemocytometer, and then re‐suspended in PBS. Cells were spiked in VERSA devices with PBS, 0.1 % BSA, and 2 mm EDTA. Percent of cells left behind in each well compared to the input number of cells was measured by placing approximately 1000 MCF7 cells in the input well. Cells were imaged in the input capture well before binding to EpCAM‐labeled PMPs in the VERSA device. Cells were counted manually.

To quantify assay sensitivity, we performed spike‐in experiments using MCF7 cells. MCF7 cells were serially diluted in PBS into approximately 5 cells, 100 cells, and 500 cells per 10 μL. To ensure accurate dilutions and to obtain a starting MCF7 cell number, 10 μL of each dilution was added to 4 glass isolator wells with PBS and Hoechst. After a 30‐min incubation, the isolator wells were imaged. Hoechst‐positive cells were counted and averaged to get a starting value for each of the 3 conditions. The 5‐cell dilution had a mean of 3.25 cells/10 μL, the 100‐cell dilution had 104.5 cells/10 μL, and the 500‐cell dilution had 658.5 cells/10 μL. MCF7s were spiked into VERSAs with PBMCs from healthy donors and processed in the VERSA according to the protocol detailed above. MCF7s were identified as being Hoechst+/Cytokeratin+/Exclusion‐. Capture efficiency percentages were determined by taking the number of MCF7s remaining after all of the wash, fixation, and permeabilization steps over the mean number of MCF7s in 10 μL of the original dilutions.