Vivotag 645

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.

The Fc3Y, Fc5X, and Fc5Y molecules were generated as described in Ortiz, 2016. Briefly, the molecules of discrete sizes and configurations were engineered using mutations based on the knobs-into-holes technology for heterodimerization and electrostatic steering for homodimerization. Placement of the homodimerization and heterodimerization Fc domains within the constructs coupled with regulated expression of the precursor peptides using separate plasmids enabled controlled assembly of Fc oligomers of different sizes and configurations. For flow cytometry analyses, constructs were labeled with VivoTag 645 (Perkin Elmer, Waltham, MA, USA) following the manufacturer’s instructions. The labeled constructs were purified by Size Exclusion Chromatography (SEC), desalted, and stored in PBS as described below. Dye loading was calculated following the manufacturer’s instructions, using extinction coefficients for the Fc3Y constructs (e~215,000 M⁻¹cm⁻¹) and the VivoTag 645 Dye (e 210,000 M⁻¹cm⁻¹).

The IL13Rα2 Ab was generated by Pfizer Inc. The fluorophores VivoTag680XL^® (VT680) (#NEV11119) and VivoTag645^® (VT645) (#NEV11173) were obtained from Perkin Elmer. AlexaFluor647^® (AF647) (#A-20006), AlexaFluor680^® (AF680) (#A-20008), AlexaFluor750^® (AF750) (#A-20011), BODIPY-X630/650^® (BOD630) (#D-10000), DyLight800^® (#46421), rhodamine-conjugated phalloidin (#R415) and Prolong antifade mounting media (#P36962) were obtained from Life Technologies, and IRDye^®800 (IRDye800) (#929-70020) was obtained from Licor. The Nunc^™ Lab-Tek^™ II Chamber Slide^™ System (#12-565-8), DAPI and an anti-human fluorescein-conjugated secondary Ab against human IgG (#PI31529) were obtained from Thermo Fisher Scientific Inc. BIOT sheep anti-human IgG polyclonal Ab (#AU003.MCUS01) and AlexaFluor647^® goat anti-human IgG (#A80-319A) were obtained from Binding Site and Bethyl Labs, respectively.