2.6 Biofloating assays
Mammalian cells were grown to 60-90% confluency, detached with trypsin-EDTA, and quenched via addition of culture medium. Dissociated cells were washed and centrifuged at 400×g for 5 min twice with PBS and stained with CellTraceTM Violet dye (Thermo Fisher Scientific). The cells were incubated with 2.5 µM dye in PBS at 1×106 cells/mL for 20 min at room temperature. Following incubation, the mammalian cells were washed three times with PBSA and then aliquoted into a 96-well plate at 5×104cells/well in a volume of 10 µL. Induced yeast cells were washed and centrifuged at 3,500×g for 3 min and resuspended in PBSA containing anti-cmyc Alexa647 antibody (Cell Signaling Technology, clone 9B11) (1:50 dilution). The cmyc-labeled yeast samples were aliquoted into the 96-well plate containing the CellTraceTMViolet-labeled mammalian cells at 10 µL/well (total volume of 20 µL/well) at a final ratio of 10:1 yeast:mammalian cells (5×105 yeast cells/well). Incubation proceeded at 4°C for 2 hr with rotation. The cells were then pelleted, washed, and resuspended in PBSA for analysis on a CytoFLEX flow cytometer. Percent bound to yeast was quantified based on the cmyc-positive fraction of the CellTraceTM Violet-labeled mammalian cells. No forward/side scatter gating was implemented.
Kinetic assays varied the incubation timepoints, wherein the 0 min timepoint sample consisted of adding the yeast to the mammalian cells and immediately pelleting and washing the sample. The yeast:mammalian cell ratio was held constant at 10:1 for kinetic assays. Titration studies were carried out by varying the yeast:mammalian cell ratios while keeping the incubation period constant at 2 hr. The percent of mammalian cells bound to scFv-expressing yeast was plotted for each ratio and fitted to a single logistic model using Prism software.