Figure 6. Flow cytometry analysis of single cell content, viability, and molecular expression.

(A) Single cells and aggregates were identified by plotting the forward scatter (FSC)-width by FSC-area for HCT 116 spheroids under different dissociation conditions. Controls received mechanical treatment by pipetting and vortexing. Single cells fall within the gated region, while aggregates are shifted to the right, and percentages are shown. (B) Cell viability was assessed by PI exclusion assay. Live cells were gated based on PI fluorescence signal, using unstained control cells as a baseline (see Supporting Information). Viability was not affected by device processing if the sample was treated first with trypsin, but did decrease significantly if treated first with EDTA or not treated at all. (C) HCT 116 spheroids were stained for the surface biomarkers EpCAM and TfR. EpCAM is a homotypic cell adhesion molecules, and expression was similar under all dissociation conditions. TfR is sensitive to trypsin cleavage, resulting in lower expression. Device treatment did not alter expression either case. (D) Similar results were observed for NCI-H1650 spheroids. MUC1 expression was also measured, and showed trypsin sensitivity. All fluorescent signals were normalized to controls that were digested with trypsin. Error represent the standard error from at least three independent experiments.