Phage Binding Quantification by ELISA and Flow Cytometry

1-step ultra tetramethylbenzidine (TMB)-ELISA substrate solution (ThermoFisher Scientific, Middletown, VA) was allowed to warm to room temperature. Cells were prepared as described in cell preparation. Cells were washed with DPBS+/1%BSA, 100 uL per well. For each type of phage and control wild-type KE phage, three wells were incubated with 40 uL of the phage (10e8 phages/uL). Phage were incubated on cells for 1 h in a humidified, 37°C, 5% CO₂ incubator. Cells were then washed three times (DPBS+/1%BSA, 100 uL) and fixed (100 uL of 2% PFA, 5 min), then washed twice more. HRP-αM13 pIII monoclonal antibody (100 uL, 1:3000 dilution in DPBS+/1% BSA, NEB) was added to each well for 1 h. Cells were washed four more times and 100 uL of TMB was added. After the TMB reacted with the HRP, the absorption was measured on a microplate reader at 650 nm. Flow cytometry was performed by binding fluorescently labeled phage (Vivotag 645, PerkinElmer, Waltham, MA [31 (link)]) to CAF or MRC5 for 30 min. Cells were washed three times and live-dead violet stain (Life Technologies) was added. Data was gathered on Beckman Coulter CyAN ADP LX and gated on cell population, live cells, and phage positive cells using FlowJo software.